Klebsiella pneumoniae (K. pneumoniae) colonizes the human gut and is a causative factor of pyogenic liver abscess (PLA). Retrospective studies conducted on K. pneumoniae PLA patients revealed subsequent CRC development in later years of their life with increasing prevalence of these strains harbouring polyketide synthase (PKS) genes. To our knowledge there are no known studies directly implicating K. pneumoniae with CRC to date. Our aims are to characterize K. pneumoniae isolates from CRC patients and investigate its effects on cell proliferation in vitro. K. pneumoniae isolates were characterized by screening virulence genes including polyketide synthase (PKS), biofilm assay, antibiotic susceptibility, and string test to determine hypervirulent (hvKp) strains. Solubilised antigens of selected K. pneumoniae isolates were co-cultured with primary colon cell lines and CRC cell lines (Stage I-IV) for 48 h. The enhancement of proliferation was measured through MTT and ECIS assay. Twenty-five percent of K. pneumoniae isolates were PKS-positive out of which 50% were hvKp strains. The majority of the isolates were from the more virulent serotype of K1 (30%) and K2 (50%). PKS-positive K. pneumoniae isolates did not possess genes to confer carbapenem resistance but instead were more highly associated with siderophore genes  and allantoin metabolism genes . Cell proliferation in primary colon, SW1116 (Stage I), and SW480 (Stage II) CRC cell lines were enhanced when co-cultured with PKS-positive K. pneumoniae antigens. ECIS revealed enhanced cell proliferation upon recurrent antigen exposure. This demonstrates the possible role that PKS-positive K. pneumoniae has in exacerbating CRC progression. Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative bacterium of the Enterobacteriaceae family that colonizes human mucosal surfaces such as the nasopharynx and the gastrointestinal tract (GIT) [K. pneumoniae has been an aetiological agent in a number of diseases such as hospital acquired infections, bacteraemia, endocarditis, pneumonia, as well as pyogenic liver abscess (PLA) [K. pneumoniae infections worldwide has risen dramatically due to the emergence of multi-drug resistant (MDR) as well as extremely drug resistant (XDR) strains [K. pneumoniae strains (hvKp) among Asian countries have also caused a peak in PLA cases being reported, especially in Taiwan and China, where K. pneumoniae has exceeded Escherichia coli (E. coli) as the most common pathogen isolated from PLA patients [K. pneumoniae (cKp) due to its increased virulence and ability to cause more severe infections compared to the latter. The hvKp strains are notorious for their biofilm forming properties as well as efficient iron uptake systems and siderophore production. These are some of the virulence factors that enhance the survival of K. pneumoniae within the host through evasion of host immune defences and its ability to sequester iron, allowing it to persist within the host. Furthermore, K. pneumoniae is known to harbour the polyketide synthase (PKS) island within its genome that produces colibactin genotoxin. This phenomenon has also been observed in E. coli, where reports demonstrated colibactin genotoxin’s ability to cleave host DNA double strands bringing about cell cycle arrest as well as DNA damage and mutations which initiates tumorigenesis [K. pneumoniae PLA patients had approximately three times higher risk of developing colorectal cancer (CRC) years after a PLA infection compared to non-K. pneumoniae PLA patients [K. pneumoniae metabolism that enter the portal venous system. [K. pneumoniae and antibiotic treatment was implemented. Furthermore, upon treating the patient with empiric antibiotic therapy (sulbenicillin 8 g/day and metronidazole 2 g/day), there was a reduction in gas accumulation due to the inhibited reproduction of K. pneumoniae. The same patient was then diagnosed with colon cancer (TNM staging: T4bN2bM0) after a few days.ct (GIT) ,2. K. pnss (PLA) ,4. In re strains . The lacpatients . Increasigenesis . This ispatients ,10,11,12 system. . The bloK. pneumoniae is a common colonizer of the GIT, especially the colon, and possibly enters the hepatic portal veins which receive blood from the GIT through the network of mesenteric veins. The inferior mesenteric vein transports blood from the colon and rectum to the hepatic portal vein which sends it to the liver before being transported back to the heart. It could also be postulated that K. pneumoniae enters the liver through this similar pathway to cause PLA.K. pneumoniae isolated from PLA patients, there are still gaps and chinks in the armour that directly link K. pneumoniae with CRC without PLA being a precursor condition. Therefore, this study aims to characterize the virulence genes as well as the prevalence of PKS-positive K. pneumoniae isolates obtained from CRC patients attending University Malaya Medical Centre (UMMC) and to determine their role in CRC progression in vitro.Although numerous studies have performed characterization of K. pneumoniae were isolated from faecal, tumour, and tissue biopsy specimens of CRC patients and healthy individuals attending UMMC upon obtaining ethical approval from UMMC Medical Ethics Committee based on the Declaration of Helsinki 1975 (Ethics Reference Number: 943.18). In this study, 43 CRC patients and 72 healthy individuals attending UMMC for routine colonoscopy screening and who had colon or rectal resection between March 2014 to August 2015 consented to participate. All clinical specimens were processed, isolated, and cultured in the Department of Medical Microbiology, Faculty of Medicine, University of Malaya. In order to isolate pure colonies of K. pneumoniae, the specimens were homogenized and cultured in Luria Bertani broth overnight and streaked onto MacConkey Agar. All mucoidal colonies were subjected for identification of K. pneumoniae isolates, performed using Bruker Biotyper MALDI-TOF MS system according to manufacturer’s instructions. Additionally, species identification was further confirmed through 16s rRNA gene sequencing. Pure colony K. pneumoniae cultures were preserved in 30% glycerol stocks and stored in −80 °C until further use.® CCL-233), SW 480 (ATCC ® CCL-228), HT 29 (ATCC ® HTB-38), HCT 116 (ATCC ® CCL-247) were used as stage I-IV derived cell lines respectively. All cell lines were grown in RPMI 1640 and supplemented with 10% Fetal Bovine Serum (FBS) and 1% each of Penicillin-Streptomycin, L-Glutamine, and Amphotericin B at 37 °C with 5% CO2.A total of 4 human CRC cell lines derived from various CRC stages (Stage I to IV) as well as 1 primary colon cell line were used in this study. Human primary colon cell  was used as a non-CRC in vitro model. SW 1116 (ATCC K. pneumoniae DNA were extracted according to manufacturer’s protocol using the Qiagen DNeasy Blood & Tissue Kit. The purity and concentration of extracted DNA were measured and quantified using the IMPLEN NanoPhotometer. The DNA purity of 1.8–2.0 (A260/A280 ratio) was used for subsequent experiments. Extracted DNA that was not utilized immediately were stored in aliquots at −20 °C. The positive and negative controls used were K. pneumoniae ATCC 13883 extracted DNA and nuclease free water respectively.K. pneumoniae virulence genes which included PKS colibactin gene, K. pneumoniae serotypes (K1(K1-magA), K2 (K2wzy), K5 (K5wzx), K20 (wzyK20), K54 (wzxK54), K57 (wzyK57)) as well as Carbapenem genes (OXA-48 (blaOXA-48), NDM-1 (blaNDM-1), KPC (blaKPC), IMP (blaIMP), and VIM (blaVIM)). Previously published primers that were used in this study along with the length of the expected PCR product are listed in mrkD1, mrkD2, fimH, kpn), Enterobactin siderophores , Ferric aerobactin receptor , Aerobactin siderophore, Yersiniabactin siderophore , Salmochelin catecholate receptor , Klebsiella iron uptake systems , Genotoxins(Colibactin (clbB), Haemolysin (hlyA), Cytotoxic necrotizing factor (cnf)), Capsule synthesis (K2 serotype capsule synthesis (k2A), Fucose capsule synthesis (wcaG), Lipid polysaccharide (LPS) synthesis (wabG), outer membrane lipoprotein (ycfM), Uridine diphosphategalacturonate 4-epimerase (uge), Regulator of mucoid phenotype A , urease synthesis (ureA), and Allantoin metabolism . The cycling conditions for the detection of PKS colibactin gene were initial denaturation at 95 °C for 10 min, denaturation at 94 °C for 45 s, annealing at 54 °C for 45 s, and extension at 72 °C for 1 min. This was repeated for 30 cycles. The amplified PCR products were separated and visualized through gel electrophoresis at 120 V for 2 h in 1.8% (wt/vol) agarose gel containing SYBR Safe DNA Gel Stain (Invitrogen). Cycling conditions used for the multiplex PCR along with the primer grouping will be made available upon request.A total of 41 primer sets were used to screen and detect K. pneumoniae isolates and 1 K. pneumoniae ATCC 13883 strain were screened against a panel of 29 antibiotics from 16 antimicrobial categories using Kirby-Bauer disc diffusion method and broth microdilution methods were based on previously established protocol by Wiegand et al. and Hudzicki et al. [A total of 40 clinical i et al. ,15 accori et al.  antibiotK. pneumoniae isolates were inoculated in 5 mL of trypticase soy broth for 24 h at 37 °C. The inoculum was adjusted to 0.5 MacFarland standard . 200 µL of the adjusted inoculum were seeded into wells of the 96-well flat bottom plates and incubated at 37 °C for 24 h. Inoculum were carefully removed, and the plates were washed three times in sterile phosphate buffered saline (PBS). The adherent layer of bacteria was heat-fixed for 20 min at 60 °C. An amount of 125 µL of 0.1% crystal violet was added to the wells and incubated at room temperature for 15 min. The plates were rinsed with sterile distilled water 3 times and excess water and dye were blotted using paper towels. An amount of 200 µL of 33% glacial acetic acid was added to the wells and incubated at room temperature for 1 h. The absorbance was measured at 570 nm and the biofilm-producing classification was determined based on the values below:This assay was performed according to previously established protocols by Singh et al. and Emery et al. ,19 with cNegative control = DOaAbsorbance value of each strain = DOa ≤ DOcNon-adherent = DOc < DOa ≤ 2xDOcWeakly adherent = DOc < DOa ≤ 4xDOcModerately adherent = 2xDOc < DOaStrongly adherent = 4xDOK. pneumoniae isolates were grown on nutrient agar plates overnight at 37 °C. A sterile inoculating loop was used to touch the surface of a single K. pneumoniae colony and to stretch the colonies by raising the inoculating loop to produce a mucoviscous string. The test was considered positive if the isolates were able to produce a mucoviscous string of > 5 mm in length.K. pneumoniae isolates were grown separately in 200 mL of Luria Bertani (LB) broth in a conical flask at 37 °C for 18 h with shaking at 180 rpm. After incubation, the grown inoculum was transferred to 50 mL conical tubes and centrifuged at 18,000× g for 30 min at 4 °C. The supernatant was discarded, and the bacterial cell pellet was washed twice in cold sterile PBS and resuspended in sterile ice-cold PBS at a concentration of 1 g wet weight per ml. The suspension was sonicated to rupture the bacterial cell wall for 15 min at 4 °C with 40 s burst at 50% power setting. The sonicated suspension was then briefly vortexed at top speed and allowed to stand overnight at 4 °C to allow complete solubilisation of the K. pneumoniae bacterial antigens. The suspension was sequentially filtered through 0.45 µm and 0.20 µm syringe filters. An amount of 10 µL of the filtered solubilised antigen was spread onto nutrient agar and incubated at 37 °C overnight to determine no viable bacterial cells were present. The whole bacterial antigens extracted are protein in nature and will be used as such. Further purification and determination of the antigen properties were not performed in this study. From here on, the K. pneumoniae solubilized antigens will be referred to as bacterial antigens. The protein concentration was estimated using the Bradford’s method and the extracts were stored in aliquots at −20 °C until further use.Single colonies of 3/mL). The cells were incubated at 37 °C with 5% CO2 overnight to allow the cells to adhere. MTT assay was performed to determine the appropriate concentration of bacterial antigen to be used (data not shown). After incubation, 10 µL of prepared bacterial antigen was added to the well at a concentration of 500 µg/mL . The optimal concentration of bacterial antigen to be used was determined through MTT assay. 10 µL of EGF at a final well concentration of 20 ng/mL and 10 µL of sterile PBS were used as positive and negative controls respectively. All conditions were in triplicates. The K. pnuemoniae bacterial antigens used were obtained from 4 main isolate groups, namely PKS-positive of CRC, PKS-positive of healthy, PKS-negative of CRC, and PKS-negative of healthy origin. The cells were then incubated for 48 h at 37 °C with 5% CO2. To measure the enhancement of cell proliferation, 10 µL of 5 mg/mL  MTT dye were added to all wells and incubated for 4 h at 37 °C with 5% CO2. After incubation, the supernatant was carefully aspirated so as to not disturb the cell monolayer and 100 µL of DMSO were added to the wells and kept on a rocking platform for 10 min to solubilise the MTT dye. The absorbance was read on a microtitre plate reader at 570 nm. The enhancement of cell proliferation was read after normalizing the values obtained from the negative control wells.Human primary and CRC cell lines were seeded into wells of 96-well flat bottom plates at a density of 500 cells/100 µL per well . The impedance was measured using the ECIS model Zθ (Z Theta) . The cells were incubated for a period of 6 days at 37 °C with 5% COp value less than 0.05 was considered to be statistically significant.The IBM Statistical Package for Social Science (SPSS) software version (26.0) was used for statistical analysis. A Pearson chi-square was used to find the correlation between the relationship of PKS-positive and hypervirulent isolates against the screened virulence genes. A K. pneumoniae. Out of the 40 clinical isolates, 25% (10/40) of the isolates harboured the PKS colibactin genotoxin gene and a K-antigen capsular serotyping screening revealed that 14/40 of the isolates could be assigned to the six most prevalent serotypes  of K. pneumoniae. The most virulent serotypes that have been associated with hypervirulence are the K1 and K2 serotype. Screening of the ten PKS-positive isolates showed that three (30%) of these isolates belonged to the K1 serotype, five (50%) were K2, one (10%) isolate was K5 and the remaining one isolate did not belong to the any of the serotypes screened. On the other hand, all three of the PKS-positive K1 serotype isolates were obtained from clinical specimens of CRC patients and the remaining seven PKS-positive isolates were obtained from healthy individuals attending UMMC for routine medical examinations. There was no statistical difference observed among the PKS-positive K. pneumoniae strains isolated from CRC and healthy individuals. Further assessment of other virulence genes is vital to understand if other virulence factors play a role in increasing the pathogenicity of PKS-positive K. pneumoniae. The presence of virulence genes was determined solely based on a PCR and sequencing of biosynthetic gene clusters will be required to determine the functionality of the virulence genes. K. pneumoniae isolates.After screening 43 CRC patients and 72 healthy individuals, only 40 individuals  harboured K. pneumoniae sequesters iron from host bound proteins through the secretion of siderophore molecules which are high-affinity iron-chelating agents. The four groups of siderophores are aerobactin, enterobactin, salmochelin, and yersiniabactin, which all play different roles in establishing an infection within the host. In this study, PKS-positive isolates harboured genes responsible for the production of aerobactin, enterobactin, and yersiniabactin siderophores. A key notable difference observed was the lack of the salmochelin  siderophore gene among PKS-positive isolates from CRC patients and healthy individuals as well as lack of iron uptake systems  among PKS-positive isolates from healthy individuals. In addition to that, PKS-negative isolates predominantly possessed genes conferring production of enterobactin siderophore. Similarly, the salmochelin siderophore was also found in approximately 17% of PKS-negative isolates from CRC patients and 11% of PKS-negative isolates from healthy individuals. Iron is an important element that is required for bacterial survival and pathogenesis. Although, iron is abundantly found within the host, most of the iron are bound to host proteins such as haemoglobin and transferrin. To overcome this, K. pneumoniae hypervirulent strains. All the PKS-positive isolates from CRC patients had the rmpA gene. Additionally, the cytotoxic necrotizing factor-1 (cnf-1) works in concert with the colibactin gene to modulate the cell cycle that contribute to tumorigenesis and hyperproliferation. This gene was also found in all PKS-positive isolates of CRC origin. However, the cnf-1 gene was not present in any of the PKS-positive strains isolated from healthy individuals and therefore it would be interesting to observe how it affects the enhancement of cell proliferation. Interestingly, the haemolysin gene (hly) was found in a low abundance of 14% and 8% among PKS-positive from healthy individuals and PKS-negative isolates from CRC patients, respectively. The two fundamental genes that are involved in colonization and infection for K. pneumoniae are the uge and urea gene. The uge gene plays a role in the initial colonization and infection of the gastrointestinal mucosa whereas the urea gene aids in driving the persistence of infection by overcoming the gastrointestinal stress through the utilisation of nitrogen for protein synthesis. The regulator mucoid phenotype gene (rmpA) is responsible for enhancing extracellular capsule synthesis resulting in increased mucoviscosity of the isolates. The presence of the rmpA gene is also one of the virulence determinants for Fimbriae and adhesins are vital for the attachment and colonization on the colonic mucosa. The kpn gene which is responsible for type 1 fimbriae-like adhesin is lacking among PKS-positive isolates from CRC patients. However, it is found in 100%, 50%, and 61% of PKS-positive , PKS-negative (CRC), and PKS-negative  isolates respectively. None of the isolates possessed the mrkD-1 gene which is a type 3 fimbriae but was compensated by the presence of other adhesins and fimbriae. Escherichia coli (E. coli) can only use allantoin as a source of nitrogen. However, K. pneumoniae has the ability to metabolize allantoin and utilize it as a source of carbon and nitrogen. This allows it to persist within the host for a prolonged time and survive under stressful conditions. Allantoin metabolism is vital for the utilisation of allantoin as a main source of nitrogen and carbon. It is also an important hallmark feature to determine the severity of infection. wabG, ycfM, entB, entB1, fimH, iutA, iutA1, iucB, rmpA1, rmpA2, kfuBC, kfu, allS, and allS2 (having a correlation significance at 0.01 level). We also observed that the presence of the pks gene is correlated with the hypervirulent K. pneumoniae strains as previously described. Additionally, these Pks-positive hypervirulent K. pneumoniae isolates possess a high correlation between the ycfM, ybtS, ybtA, (yersiniabactin) iutA, iutA1, aerobactin, iucB, (aerobactin) rmpA1, rmpA2 (regulator of mucoid), and k2A (K2 capsule) genes.A correlation analysis was performed to determine the relationship of PKS-positive and hypervirulent isolates against the screened virulence genes . The anaAntibiotic resistance is a worldwide threat, particularly in clinical settings where growing cases of MDR, XDR, and Pandrug resistance (PDR) infections are being reported. With the rise of hvKp strains being reported in Asian countries it is important to contain the spread of these isolates and prevent the emergence of MDR-hvKp strains prevailing. An isolate is considered to be MDR when it is non-susceptible to ≥1 agent in ≥3 antimicrobial categories. An XDR isolate is defined as non-susceptible to ≥1 agent in all but ≤2 categories and a PDR isolate is non-susceptible to all antimicrobial agents .K. pneumoniae isolates and one K. pneumoniae ATCC 13883 strain, seven clinical isolates were identified as MDR strains, all of which were PKS-negative. PKS-positive K. pneumoniae isolates were only resistant to the penicillin group of antibiotics (ampicillin (6/10) and amoxycillin (10/10)). On the other hand, PKS-negative K. pneumoniae conferred antibiotic resistance to amoxycillin (30/30), ampicillin (27/30), chloramphenicol (6/30), tetracycline (4/30), trimethoprim-sulfamethoxazole (4/30), doxycycline (3/30), levofloxacin (3/30), tigecycline (2/30), amikacin (1/30), amoxicillin-clavulanic acid (1/30), and norfloxacin (1/30). AST results are tabulated in After screening fourty clinical blaOXA-48 gene. Of these strains, two out of three were isolated from healthy individuals and one strain was isolated from a CRC patient. The summary of isolates harbouring carbapenem resistance are shown in Additionally, none of the PKS-positive isolates carried the genes that confer resistance towards carbapenems. However, three PKS-negative isolates carried the K. pneumoniae biofilm-forming capabilities are a cause of the majority of indwelling catheter infections among patients. In this study, 18 out of the 40 isolates screened were strong biofilm producers. Among this group, five isolates were PKS-positive strains and out of which three isolates were obtained from CRC patients. There were six moderate biofilm producers with two PKS-positive isolates, one of which came from a CRC patient. Four isolates were weak biofilm producers having only one PKS-positive isolate obtained from a healthy individual. The remaining 12 out of the 40 isolates screened were non-biofilm producers out of which two PKS -positive isolates were obtained from healthy individuals. K. pneumoniae isolates.K. pneumoniae that the strain is considered to be a hvKp strain [K. pneumoniae isolates screened, 16 (40%) produced a positive string test. In these 16 isolates, 10 isolates (62.5%) were considered hvKp based on the abovementioned definition. The further breakdown of the hvKp isolates is described in K. pneumoniae in CRC is similar to the healthy group, with 34.8% and 34.7% prevalence, respectively  wells. The enhancement of cell proliferation was expressed as the percentage (%) of cell proliferation. Generally, K. pneumoniae antigens were able to enhance the proliferation of human primary and CRC cell lines in vitro.However, PKS-positive isolates isolated from CRC patients had the highest enhancement of cell proliferation across all cell lines except for SW1116 (stage I), where the PKS-positive isolate from healthy patients had a slight edge. The highest enhancement of cell proliferation was observed in the SW480 (stage II) cell line. The enhancement of cell proliferation trend showed a bell-shaped curve that progressed from the human primary cell line and peaked at SW480 (stage II) after which the enhancement of cell proliferation decreased. Additionally, PKS-positive isolates showed better enhancement of cell proliferation compared to PKS-negative isolates, irrespective of isolate origin. There is some plausibility that the presence of the PKS colibactin gene along with co-virulence factors could play a role in enhancing the progression rate of CRC, especially in the normal and early stages of CRC (as observed in the primary and CRC cell line of stage I and II).K. pneumoniae antigens. In this section only, results of the cell lines exposed to PKS +ve CRC and PKS -ve CRC antigens were shown as it had the best enhancement of its group. The enhancement of cell proliferation was seen in all stages of CRC cell lines. Interestingly, the enhancement was more pronounced upon the second exposure of K. pneumoniae antigens. Recurrent exposure to K. pneumoniae antigens simulates the persistent exposure of the colonic mucosa to bacterial toxins within the colonic microenvironment. The enhancement of cell proliferation due to recurrent infection by K. pneumoniae antigens demonstrates the effect that chronic K. pneumoniae infection has on cell proliferation and possibly initiation of tumorigenesis. PKS +ve CRC had a higher cell proliferation rate at the early stages of CRC  whereas PKS-ve CRC antigens enhanced cell proliferation at advanced CRC stages (HT29 and HCT116). These findings could potentially illustrate the role of PKS +ve K. pneumoniae as a driver bacterium involved in the initiation of tumorigenesis and PKS-ve K. pneumoniae as the passenger bacterium that progresses the tumorigenesis into more advanced stages of CRC.The ECIS array aids in detecting an increase in cell proliferation by measuring the impedance of the cell when an alternating current is applied to it. As the cell proliferates and covers the electrodes at the bottom of the well, the impedance increases. K. pneumoniae infections are a huge threat and burden to the healthcare systems as they are the leading cause of nosocomial infections. The emergence of hvKp strains further complicates the treatment and prognosis of a patient. In this study, there was an equal distribution of hvKp strains from PKS-positive and PKS-negative K. pneumoniae isolates as well as from CRC and healthy individual origin. Although hvKp strains are generally sensitive to most antibiotics, it is still more difficult to treat compared to cKp infections due to the arsenal of virulence factors that hvKp isolates possess [OXA-48 gene. Globally, the K1 and K2 serotype are the most prevalent serotypes of the hvKp strain and PKS-positive K. pneumoniae isolates. This could possibly explain the higher rate of antibiotic susceptibility and lack of antibiotic resistance genes observed among PKS-positive strains in this study. The K1 and K2 serotype are known to have increased capsule production that confers the hypermucoviscous variant. This characteristic presents a physical barrier that prevents the horizontal gene transfer of antibiotic resistance genes to occur [K. pneumoniae isolated from PLA patients is rampant and should be addressed. possess . We obseto occur . Howeverto occur ,25. Therto occur . The incK. pneumoniae, three of which were isolated from CRC patients. Moreover, the characterization of isolates based on virulence genes also showed that these three isolates possessed 22/28 of the virulence genes screened in addition to being a strong biofilm producer and belonging to the K1 serotype. These features aid in increasing their virulence, thus making them strong pathogenic bacterium with the ability to cause life threatening and debilitating diseases within the host.There has been plenty of controversy surrounding the definition of hvKp strains through string test . Hence, K. pneumoniae isolates is the abundance of virulence genes present within the PKS-positive K. pneumoniae genome. PKS-positive isolates had a greater abundance of genes responsible for siderophore production, namely aerobactin, enterobactin, and yersiniabactin, compared to PKS-negative isolates that were more associated with only enterobactin siderophore. The ability to produce more than one group of siderophore increases the organism’s ability to sequester iron more efficiently from the host environment, thus enhancing its survival within the host. A similar observation was made by Khaertynov et al. [rmpA-positive isolates.The key observable difference between the PKS-positive and PKS-negative v et al. , where aK. pneumoniae isolates that harbored a single type of siderophore, particularly enterobactin, had an increased virulence compared to isolates that harbored all four types of siderophore production genes. Similarly, PKS-negative isolates in this study primarily had genes for enterobactin siderophore production only, which translated to having a higher antibiotic resistance thus producing MDR strains. However, this does not diminish the pathogenicity of PKS-positive strains as they are primarily implicated in invasive and persistent infections as well as their increased ability to cause severe infections such as PLA, brain abscess, ophthalmitis, and bacteraemia that results in sepsis [A study by Chen et al.  demonstrn sepsis .allS, allS2) was more expressed in PKS-positive strains. Allantoin metabolism is a vital process that allows an organism to utilize allantoin as a source of carbon and nitrogen. Chou et al. [K. pneumoniae was able to utilize not only carbon but nitrogen as a source of energy under both aerobic and anaerobic conditions. This shows the ability of PKS-positive isolates to sustain and persist under stressful conditions and environments such as the tumour microenvironment within a CRC patient. The results of the virulence gene detection are listed in Additionally, the genes coding for allantoin metabolism  that may induce inflammation resulting in increased cell proliferation. This indicates that the colibactin toxin production along with its counterpart virulence genes seen in the PKS-positive isolates has a better ability to enhance CRC progression compared to PKS-negative isolates. The systematic review conducted by Strakova et al. [K. pneumoniae as a driver bacterium inducing mutations in CRC driver genes. As observed in K. pneumoniae antigens, with the highest proliferation occurring in SW 480 (stage II) CRC-derived cell lines. Contrarily, CRC cell lines exposed to PKS-negative K. pneumoniae antigens had a greater enhancement of cell proliferation at advanced CRC stages (stage III-IV). The findings from this study could possibly indicate the driver–passenger relationship between PKS-positive and PKS-negative K pneumoniae, respectively. To further explore this notion, an ECIS assay was carried out to substantiate these findings.To further understand if other virulence factors and the origin of isolate may play a synergistic role in PKS-positive isolates, we co-cultured human primary and CRC cell lines derived from stages I-IV with a et al.  discusseK. pneumoniae antigens. Repeated exposure of bacterial antigens increased the enhancement of cell proliferation significantly after the second antigen introduction. This could be due to the medium renewal as well. However, as seen in K. pneumoniae as a driver bacterium in the initiation and progression of CRC. Another interesting pattern observed here was the ability of PKS-ve CRC-exposed cells to sustain proliferation over longer periods of time compared to PKS+ve CRC-exposed cells. Although we may not see spikes in impedance of cells exposed to PKS-ve CRC cells, they are able to demonstrate a gradual increase in proliferation which will be useful in sustaining the progression of CRC cell proliferation. This is supportive of the notion that PKS-ve K. pneumoniae is a passenger bacterium.The ECIS array was used to confirm the enhancement of CRC cell growth upon exposure to K. pneumoniae isolates in possibly enhancing CRC progression in vitro. However, a more in-depth molecular study should be performed to understand the various aspects affected by virulence gene expression during tumorigenesis. Our observation suggests that the presence of the PKS gene and its counterpart virulence genes do play a role in enhancing CRC progression as summarized in K. pneumoniae strains is vital to dampen the progression of the tumor while improving the management of CRC patients.To our knowledge, this is the first known study to highlight the effect of PKS-positive


The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model.The online version contains supplementary material available at 10.1007/s00018-022-04557-y. Metastasis is the main cause of cancer-associated mortality and one of the most indicative prognostic factors for cancer patients –4. This The Integrin alpha9 (ITGA9) protein is composed of the α9 itself and a β1 subunit. Unlike other integrins, ITGA9 does not depend on the Arg-Gly-Asp (RGD) sequence for ligand recognition  and bindOne of the most remarkable families of ITGA9 interactors is the ADAM family, which has some members highly expressed in many types of cancer and can regulate cell–cell adhesion or cell-ECM interaction through its binding to adhesion molecules such as integrins and syndecans . Overall2 and were periodically tested for mycoplasma by PCR and authenticated by short tandem repeat profiling.RMS cell lines RH30 and RD were cultured in Minimum Essential Medium with Earle’s Salts (Biowest), NB cell lines BE(2)-C and CHLA90 were grown in Iscove’s Modified Dulbecco’s Medium (Thermo Fisher Scientific) and BC cell lines MDA-MB-231 and MDA-MB-468 were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific). All media were supplemented with 10% FBS (Sigma), 1 × non-essential amino acids, 100U/ml penicillin and 0.1 mg/ml streptomycin . All cell lines were maintained at 37 °C in a controlled atmosphere of 5% CO5 target cells were seeded in 60 mm dishes and incubated together overnight. Positively transduced cells were selected with puromycin . Four different shRNAs were tested and the ones that reduced ITGA9 expression more efficiently were selected for further experiments. Rescue experiments to overexpress ITGA9 were performed using pcDNA3.1(+) vector (GenScript Biotech), carrying ITGA9 complete cDNA (NM_002207.3). Cells were transiently transfected with 2 μg of each construct using Lipofectamine (Thermo Fisher) and protein analyses were performed after 24 h.Genetic ITGA9 downregulation was performed by shRNA cloned into the lentiviral vector pGIPZ . Lentiviruses were produced in HEK293T cells and 2 × 10At 80%-confluence, cells were washed with PBS and scraped in RIPA protein lysis buffer (Thermo Fisher Scientific) supplemented with protease (Roche) and phosphatase inhibitors (Sigma-Aldrich). Cell lysates were incubated for 5 min at 95 °C and after centrifugation at 13000 rpm for 15 min at 4 °C, cell debris was discarded. Supernatant protein concentration was quantified by the DC Protein Assay (Bio-Rad) according to manufacturer’s instructions, and 20-40 µg of total protein was separated on 8–12% SDS-PAGE gels and transferred onto PVDF membranes . After blocking with 5% BSA (Sigma-Aldrich) for 1 h, membranes were incubated overnight at 4 °C with the following primary antibodies: anti-ITGA9 , anti-FAK , anti-pFAK at Tyr397  and anti-actin . Finally, membranes were incubated with the corresponding HRP-conjugated secondary antibodies for 1 h at room temperature and immunoreactive bands were visualized with ECL Prime reagent .5 cells resuspended in serum-free medium were seeded in the upper chamber of a Matrigel-coated Transwell (Corning), and the lower chamber was filled with serum-supplemented medium. Cells were incubated at 37 °C for 24 h and fixed with 4% paraformaldehyde (Sigma-Aldrich). After removal of the Matrigel, the cells that migrated to the lower surface of the membrane were stained with Hoechst-33342  and were counted under the microscope. Three independent wells were counted for each condition. To assess RA08’s effects on cell invasion (and RGD peptide), cells were previously treated for 48 h with different concentrations , which were also present in the medium during the cell invasion assay.1 × 10Cell proliferation was measured using crystal violet staining. Cells were seeded at low-density in 96-well plates, and a different initial number of cells were used for each cell line to ensure lineal growth throughout the assay. Media was changed every 48 h, including RA08 (or RGD peptide) treatment . Cells were allowed to proliferate for 5 days and were washed with PBS, stained with 0.5% crystal violet (Sigma-Aldrich) and washed thoroughly with distilled water. Plates were dried overnight and crystals were dissolved with 15% acetic acid (Carl Roth). Finally, absorbance was measured at 590 nm using an Epoch microplate spectrophotometer (Biotek).Anchorage-independent cell growth was measured by WST-1 (Sigma-Aldrich), following the manufacturer’s instructions. Cells were seeded at low density in polyHEMA-coated dishes (Santa Cruz Biotechnology) that prevented cell attachment to the plate and were allowed to grow for 48 h. At the end of the assay, cell spheres were disaggregated with accutase (Fisher Scientific) and WST-1 reagent was added to the medium. Three hours later, absorbance was measured at 440 and 690 nm using an Epoch microplate spectrophotometer (Biotek).6 RD cells, 1.25 × 105 MDA-MB-468 cells, and 1 × 104 and 2 × 104 BE(2)-C cells for the shRNA and RA08 models, respectively). To test RA08 effects, mice were split into three groups of 10 mice each and treated with the vehicle  or two different RA08 doses by subcutaneous injection. The first RA08 administration was performed two hours before tail vein injection and then three times per week throughout the experiment. Mice were monitored and their body weight measured twice per week. Ethical endpoint criteria were based on: acute weight loss , clear detection of metastases (lumps or swelling) or poor general appearance of the animal. Metastatic foci were found in post-mortem analysis of mice fulfilling at least one of these end-point criteria. After euthanasia, all organs were assessed and macroscopic metastases were detected. Then, tissue samples were collected, fixed in formalin (Sigma-Aldrich) and paraffin-embedded. In the case of RMS models, most of the metastases were found in lungs, ovary and suprarenal gland, while in the NB models the majority of metastases were hepatic. For the BC model, lung sections were deparaffinized and rehydrated for incubation with anti-GATA3 antibody . All procedures were approved by the Ethics Committee of Animal Experimentation of the Vall d’Hebron Research Institute (CEEA) and were in line with EU directive 2010/63/EU.Tumour cells were intravenously injected through the tail vein of five-week-old SCID-beige mice (Charles River Laboratories) housed in specific pathogen-free conditions. Cell number was adjusted according to the cell line’s ability to generate metastases  was purified in a semipreparative system equipped with a NW50 Merck column filled with 10 µm of kromasil silica, and the pure fractions analysed in an analytical RP-HPLC were lyophilized. The ion-exchange step was carried out to obtain the peptide with the acetate counterion. The final acetate compound was recovered via filtering and then lyophilized. (13.4 g were obtained.) RA08 was characterized by mass spectrometry by ESI–MS spectrometry (MW = 1806 a.m.u.).RA08 was synthesized by solid-phase peptide synthesis following the Fmoc/tBu strategy with 4-Methylbenzhydrylamine (p-MBHA) resin at the 30 mmol scale. Fmoc-AM-OH linker (2 eq.), HOBT (2 eq.) and DIPCDI (2 eq.) were used for attaching the linker to the resin. Then, synthesis proceeded with the removal of the Fmoc group by treatment with a solution of 20% piperidine in DMF (twice for 5 min and twice for 10 min). The peptide-resin was washed 5 times with DMF, filtered and the amino acids were coupled. Couplings were performed by using Fmoc-AA-OH:DIPCDI:HOBt (3 eq: 3 eq: 3 eq) in DMF (Fmoc-Msa-OH was coupled using 1.5 eq). All these coupling reactions were left to react for 40–60 min. The completion of each coupling reaction was controlled with a ninhydrin test (or a Chloranil test). After completion of the peptide sequence, the peptidyl-resin was washed with DMF, MeOH and Et2O and dried. 92.6 g of peptidyl-resin were obtained. Then, the peptidyl-resin was left to react for 2 h in a TFA mixture (TFA:TIO:TIS:H2O (85:5:5:5) ) at room temperature to cleave the peptide from the resin. The resin was washed with TFA and EtThe binding of RA08 to ITGA9 was assessed using the method of the cellular thermal shift assay (CETSA) as previously described . BrieflyLZerD protein docking suite [−1). The ADAM-12/ITGA9 binding affinity was also calculated with the same web server. All analyses were performed from protein structure predicted by homology-modelling server SWISS-MODEL [In silico analysis of RA08 binding to ITGA9 was performed using the platform ng suite  to preding suite  and calcSS-MODEL .t test or one-way ANOVA , considering *p < 0.05, **p < 0.01 and ***p < 0.001. Log-rank test was used for survival analysis.At least three independent replicates were performed for each experiment and analysis was performed using GraphPad Prism Software. Error bars in graphical data represent mean ± SD. Statistical significance was determined by Student’s ITGA9 expression was downregulated in cells stably transduced with two specific shRNAs sh#1 and sh#2) both very effective in reducing the amount of protein compared with empty vector-transduced cells (CTRL). Western blots -C, were selected to perform non-adherent cell growth assays and subsequent in vivo studies. Cells overexpressing sh#1 and sh#2 seeded in non-adherent conditions showed a reduction in cell proliferation after 48 h  a higher ITGA9 expression was observed in relapsed patients when compared with the subgroup without relapse  derived from the disintegrin domain of the native sequence of ADAM-12 (-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-). RA08 has been modified at the N-terminus with the inclusion of an unnatural amino acid , together with other modifications such as the substitution of Cys by Met, the substitution of Phe by 3-mesitylalanine , and the incorporation of two Lys at the N- and C-termini of the sequence. All these modifications aim to increase its stability in plasma and improve its pharmacological properties for inhibiting the ITGA9-ADAM interaction, with the ultimate aim of impairing cancer cell invasion and metastasis. Additionally, the incorporation of two further Lys residues, one at the N-terminus and another at the C-terminus, was performed to improve the peptide sequence’s solubility when formulated in physiological saline  in RMS and NB cell lines. In this case, since it has been previously suggested that ITGA9 played a role in breast cancer (BC) progression, BC cell lines were also included to test the possibility of the compound in this neoplasia. The results showed a remarkable reduction in FAK phosphorylation in RA08-treated cells in a dose-dependent manner, whereas total FAK and ITGA9 levels were unaltered Fig. b to e.p value: 0.016), observable from 55ºC onward, thereby suggesting that RA08 binds ITGA9 and protects it against thermic degradation  between beta-propeller domain for ITGA9 and the domain of ADAM12 that contains the peptide that inspired RA08. For more information and interactive format of the 3D structure pbd files, see Supplementary File 1 (RA08/ITGA9) and 2 (ADAM12/ITGA9).Additionally, the in silico analysis revealed also a high probability of binding between RA08 and ITGA9 (on its beta-propeller domain) with a binding affinity prediction value of − 13.4 Kcal molTreatment with RA08 produced a significant reduction in cell invasiveness in the majority of RMS, NB and BC cell lines, with RA08 concentrations ranging from 100 to 1000 nM. The effects of RA08 were tested in two cell lines for each of the cancers included in this work Fig. a. In RMSSimilarly, treatment with RA08 reduced cell growth when cells were cultured in anchorage-independent conditions, concurring with the previous results obtained with the genetic depletion of ITGA9. Thus, in the 3 cell lines analysed, a reduction of ~ 30% in anchorage-independent cell growth was observed in the presence of RA08 at 1000 nM Fig. b.With the aim of evaluating the therapeutic potential of RA08 in vivo murine metastatic experimental models were established by tail vein injection. In the RMS model Fig. a, all coLikewise, the NB model Fig. c revealeFinally, the BC model showed a diffuse pattern of invading cells and it was not possible to quantify the number of macroscopic lesions. In this case, with the aim of evaluating metastatic foci in mouse lungs, immunohistochemical staining of GATA3 was used as a marker of tumour cells. Although the quantification procedure was different due to the diffuse nature of the metastatic cells, the results also showed a reduction in metastatic foci to half for the two doses analysed (1 mg/kg and 2 mg/kg) Fig. e and f.It is well known that the onset of metastasis very often involves crossing a boundary beyond which the probability of cure becomes clearly lower. The fact that most of cancer-related deaths are attributed to metastases, but, unfortunately, treatments for their blockade have not yet been established, is a tremendous paradox. Probably, the more advanced studies in this direction were focused on inhibiting angiogenesis , 37, matA noticeable variable of metastasis is its temporal course, which strongly depends on cancer type. For instance, in small cell lung cancer and pancreatic cancer, metastatic disease often appears at the time of (or shortly after) the initial diagnosis, whereas many patients with breast and prostate cancers, among many others, tend to show metastasis after a prolonged period of time . These oMYCN-amplified in NB) are completely independent of ITGA9 levels. Conversely, in the case of patients classified as fusion-negative in RMS and non-MYCN amplified in NB, the outcomes showed dependency on ITGA9 levels. Consequently, a blocking of its function may hinder the development of metastasis in these specific subtypes. Interestingly, fusion-negative RMS and non-MYCN-amplified NB are considered to have better prognosis compared with less favourable subgroups of patients; however, unresolved questions persist regarding the optimal management for these groups of patients given that some cases can still relapse and/or metastasize. The fact that, in RMS and NB, ITGA9 can discriminate a subgroup of patients with worse prognosis should be considered for subsequent studies, as this new set of patients with worst prognosis would be candidates for putative ITGA9-based anti-metastatic therapy or, at least, for an intensification of treatment owing to their putative propensity to metastasize. Two BC cell lines were included in this part of the study to test the possible applicability of the compound to other neoplasia. In these cell lines, variability was also observed between the 2 cell lines analysed, with the cell line MDA-MB-468 showing significant sensitivity to the compound whilst in the other (MDA-MB-231), effects were only observed at the highest dose (1000 nM). Both cell lines correspond to triple negative BC, but relative ITGA9 expression is notably higher in MDA-MB-468 , thereby suggesting that the expression level may correlate with sensitivity to treatment with RA08.The experiments herein provided revealed a strong dependency on ITGA9 in several steps of metastasis, even though the experimental tail vein models provided skip initial steps of the process and, therefore, ITGA9 role may be even more extensive. The RGD peptide used as a reference promoted similar effects on cell invasiveness. However, the strong concomitant reduction observed in cell growth pointed to a higher toxicity and to a less-specific activity, probably caused by the wide range of integrins and ligands potentially affected by RGD blocking. This reinforces our approach and suggest a greater specificity of ITGA9 blocking as well as lower toxicity. Interestingly, a strong variability in the response to the inhibition of ITGA9 among different cell lines was detected. Particularly, RD and BE(2)-C cell lines were clearly more sensitive than CHLA90 (slightly sensitive), or RH30 (moderately sensitive). These differences observed among cell lines pointed to a diversity of response probably caused by the molecular background of each cell line, ITGA9 basal expression  and the histology of the tumours from which were derived. Interestingly, the patient’s gene expression data presented , although clear effects upon reduction of metastasis were found in RMS, NB and BC in vivo models. Beyond the effects of RA08 on metastasis, the low toxicity of the compound makes it particularly interesting, since a putative therapy for metastasis prevention is expected to be long-term, probably spanning several months and, therefore, its possible future implantation is going to be enormously facilitated if the compounds used show a complete absence of associated non-desirable side effects.Taken together, our results indicate an important role of ITGA9 in the establishment of metastasis and present RA08 as a new compound able to prevent its origin in RMS and NB models, with potential applicability in a wide range of tumour types. In addition, the prognostic value of ITGA9 expression is also depicted, emphasizing the potential of this protein as prognostic factor in some subsets of patients.ITGA9 is expressed in RMS, NB and BC cell lines. Representative immunoblot showing ITGA9 protein levels in a RMS, b NB and c BC cell lines (PDF 845 KB)Comparative gene expression analysis of alpha integrins in RMS by its molecular subtype (fusion-positive (FP+) vs fusion-negative (FN-)). Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance  was obtained after two-tailed t-test (PDF 71 KB)Comparative gene expression analysis of alpha integrins in RMS FP+ by its state . Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance  was obtained after one-tailed t-test (PDF 46 KB)Comparative gene expression analysis of alpha integrins in RMS FN- by its state . Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA3, ITGA11, ITGAD and ITGAX. Statistical significance (*p < 0.05) was obtained after one tailed t-test (PDF 54 KB)Gene expression analysis of alpha integrins in NB MYCN amplified compared to MYCN non-amplified. Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. Statistical significance  was obtained after two-tailed t-test (PDF 155 KB)Gene expression analysis of alpha integrins in relapsed NB MYCN non-amplified compared to non-relapsed. Scatter dot plots representing the gene expression for each sample. Lines and error bars represent the mean ± CI 95%. No data was available for ITGA11 and ITGAD. Statistical significance (*p < 0.05) was obtained after one-tailed t-test (PDF 57 KB)Relapse-free survival curves of NB MYCN non-amplified patients considering each alpha integrin expression. For each alpha integrin analysed, samples (n = 102) were separated in high or low expression considering the median gene expression. Statistical significance was obtained after Log-rank test to compare survival distributions (PDF 158 KB)Supplementary file8 (PDF 110 KB)Relapse-free survival curves of NB MYCN non-amplified patients considering each alpha integrin expression. For each alpha integrin analysed, samples (n = 102) were separated in high or low expression (PDF 11810 KB)Treatment with RGD peptide significantly reduces cell invasiveness and cell growth. a. Relative cell invasiveness and cell proliferation after treatment with increasing concentrations of RGD peptide  in RMS (RD and RH30), NB (BE(2)-C and CHLA90) and BC (MDA-MB-468 and MDA-MB-231). Bars represent cell invasion (significance indicated with asterisks), whereas cell proliferation is represented by dots and lines (significance indicated with hashes). Representative images of the Transwell-invasion assays, including control cells and cells treated with 1000nM are also shown below each plot. All measurements were taken in triplicates. All values are expressed in percentages and referred to those of the control condition. Statistical significance  was obtained after one-way ANOVA test. b. Molecular structure, name and chemical formula of the RGD peptide used for this experiment (PDF 5713 KB)File containing the 3D structure of RA08 and ITGA9 predicted interaction from LZerD protein docking suite platform (PBD 655 KB)File containing the 3D structure of ADAM12 and ITGA9 predicted interaction from LZerD protein docking suite platform (PBD 1174 KB)Below is the link to the electronic supplementary material.


Merkel cell carcinoma (MCC) is a rare but highly aggressive skin cancer. Despite important progress, overall understanding of the events that drive MCC carcinogenesis remains incomplete. We discovered that the plasma membrane citrate transporter (pmCiC) is upregulated in Merkel cell carcinoma cell lines. Cancer cells import extracellular citrate via pmCiC to support their metabolism, which is critical to support proliferation and metastatic spread. In this study, we show that inhibition of pmCiC can decrease the growth rate of Merkel cell carcinoma cell lines. Targeting pmCiC and thereby the tumor metabolism should be considered further as a potential anti-cancer therapy.+-gluconate) blocks this citrate-induced proliferation. Furthermore, the 3D in vivo Chick Chorioallantoic Membrane (CAM) model showed that the application of Na+-gluconate also decreases Merkel cell carcinoma growth. Based on our results, we conclude that pmCiC and extracellular citrate uptake should be considered further as a potential novel target for the treatment of Merkel cell carcinoma.Merkel cell carcinoma (MCC) is a rare but highly aggressive tumor of the skin with a poor prognosis. The factors driving this cancer must be better understood in order to discover novel targets for more effective therapies. In the search for targets, we followed our interest in citrate as a central and critical metabolite linked to fatty acid synthesis in cancer development. A key to citrate uptake in cancer cells is the high expression of the plasma membrane citrate transporter (pmCiC), which is upregulated in the different adenocarcinoma types tested so far. In this study, we show that the pmCiC is also highly expressed in Merkel cell carcinoma cell lines by western blot and human tissues by immunohistochemistry staining. In the presence of extracellular citrate, MCC cells show an increased proliferation rate in vitro; a specific pmCiC inhibitor (Na Merkel cell carcinoma (MCC) is an aggressive tumor of the skin with a rising incidence  that preAfter the diagnosis of MCC, a complete excision (1–2cm margin) and adjuvant radiation is normally performed. Sentinel lymph node mapping and biopsy are recommended together with stage imaging , or positron emission tomography (PET)) . Around Membrane transport proteins are involved in the movement of ions and small molecules across biological membranes. Many different membrane transporters have been shown to facilitate cancer development, progression, and drug resistance . A recenIn this study, we have conducted experiments to explore the hypothesis that pmCiC is important for MCC tumors by first measuring expression in MCC cell lines and human tumor biopsies. We then tested the effects of extracellular citrate on MCC proliferation in vitro and applied gluconate-based pmCiC inhibition to these in vitro and in vivo systems to show whether the tumor promoting effects of citrate can be inhibited.We first tested whether characterized MCC cell lines express pmCiC in vitro. All four MCC cell lines  showed high expression of pmCiC by Western blotting A.Next, we utilized immunohistochemical staining to detect pmCiC expression in formalin-fixed and paraffin-embedded primary MCC tissue sections. These results showed that 26 of the 28 samples stained positive for pmCiC (93%), where 2 samples showed no pmCiC expression (−), 9 showed weak/partial expression (+/−), 9 showed high expression (+), and 8 had very high expression (++) B. In totp = 0.00036; 72 h: p = 0.00007; t-test with citrate 48 h: control p = 0.0017; gluconate p = 0.0008; citrate + gluconate p = 0.0007). After adding 100 µM Na+-gluconate to inhibit pmCiC-mediated citrate transport into cells, the proliferative effects of citrate were abrogated. Notably, Na+-gluconate treatment alone had no effect on PeTa MCC cell proliferation .To investigate the effect of citrate on MCC cells, we used a dialyzed serum that does not contain citrate in experimental cultures of MS-1 cells. For this experiment, one group of cultures was supplemented with 200 µM citrate and the proliferation rate in both groups was measured after 24, 48, and 72 h using the CyQUANT Cell Proliferation Assay. There was a significant difference at 48 and 72 h between cells treated with 200 µM Na+-citrate versus cultures without added citrate,  decreased tumor growth when treated with Na+-gluconate externally for 5 days compared to tumors treated with NaCl (+-gluconate (concentration 500 mg/kg/d) or NaCl was applied every 24 h, as previously described (n = 20) [+-gluconate , as well as for WaGa . These results suggest not only a direct effect of gluconate on proliferation, but also on angiogenesis.Bhat et al. showed that the Chick Chorioallantoic Membrane (CAM) model works well for in vivo testing of MCC tumor cell development . In our ith NaCl B. Na+-gl(n = 20) . Consistluconate C and macol group D. This e+-gluconate as a specific pmCiC inhibitor. Growth inhibition was most probably due to cell cycle inhibition, rather than induction of apoptosis. We validated these findings using the 3D in vivo CAM model, where Na+-gluconate lowered MCC proliferation and showed antiangiogenic effects on tumor-surrounding vessels. Therefore, our study supports further research to therapeutically target the pmCiC in human MCC.In our present study, we primarily provide evidence that citrate and pmCiC expression may play a role in the biology of human MCC. PmCiC is expressed in 93% of MCC tissue samples and in 4 out of 4 tested cell lines. In vitro, we could detect an effect of extracellular citrate on the proliferation rate of PeTa and MS-1 cell lines. This effect could be blocked in PeTa cells by adding NaPmCiC expression in MCC human tissue was present in most of the specimens studied. Due to the incomplete clinical patient history available, it is difficult to make a firm conclusion about the potential correlation of staining intensity with disease outcome. However, our data strongly suggests that pmCiC is expressed in most patients with metastatic disease, which is consistent with our hypothesis that extracellular citrate uptake plays an important role in supporting metastatic behavior in vivo . From a Citrate is the primary substrate in fatty acid synthesis, which is a metabolic hallmark of cancer . By exte+-gluconate as a specific pmCiC blocker [+-gluconate treatment correlates with the lower MCC tumor cell proliferation rate when deprived of citrate [Blocking of citrate uptake by cancer cells using Na blocker  reduced  blocker . The cle citrate . Glucona citrate ,24. Mech citrate .2O2 for 10 min, samples were washed once with bi-distilled water, heated in HIER Citrate Buffer pH6  at 90 °C for 20 min, and cooled for 20 min. Samples were blocked using the blocking solution of the ZytoChem Plus HRP-kit Rabbit/Mouse (Zytomed) for 10 min.Paraffin-embedded sections were dewaxed using xylol  and then rehydrated by washing twice with absolute ethanol, twice with 96% ethanol, and twice with 70% ethanol. After blocking endogenous peroxidases using 3% HSections were labeled at 4 °C overnight using a rabbit monoclonal antibody against pmCiC  and a rabbit monoclonal antibody against Ki67 . After washing with DPBS, sections were incubated with biotinylated secondary anti-rabbit (HRP060-RB) antibodies for 30 min, washed in DPBS, and incubated with streptavidin HRP conjugate for 20 min . After washing with DPBS, slides were stained using AEC+ High Sensitivity Substrate Chromogen Ready-to-Use , counterstained using hematoxylin , and mounted using Aquatex (Merck). Tissues were collected from patients of the department of dermatology at the university hospital of Regensburg and correlated with clinical outcome. Ethics permission No. 22-2834-104.+- gluconate , and dialyzed serum . The following antibodies were used: pmCiC specific antibodies  and Ki67 . Experimental media consisted of glucose free RPMI-1640 , 10% dialyzed serum, 2 mM glutamine, 0.25 g/L glucose, ±200 µM citrate, and ±100 µM Na+-gluconate, unless otherwise stated. The incubation time varied between 24 h, 48 h, 72 h, and 5 weeks, as specified. Western blots were analyzed by measurement of the pixel density using ImageJ software , University of Wisconsin, Madison, WI, USA). The CyQUANT Direct Cell Proliferation Assay  was used to measure cell proliferation. Cells were counted with LUNA-FL™ Automated Fluorescence Cell Counter . Western blots were analyzed by measurement of the pixel density using ImageJ software.Cell lines from primary Merkel cell carcinoma (PeTa), as well as from metastatic disease , were grown in RPMI-1640 medium with 1% penicillin/streptomycin, 1% L-glutamine, and 10% fetal bovine serum. Although cell lines were not further authenticated, they were grown at low passage numbers from original sources and were kept typically in culture for only 2 months. Cells were tested and confirmed to be mycoplasma free. The following chemicals were used: citric acid and Na6 Merkel cell carcinoma cells (WaGa and MKL-1), daily treatment with either Na+-gluconate (500 mg/kg/d) or NaCl 0.9% was performed (n = 20). Pictures were taken and tumors were removed after 5 days. The size of tumors was measured and the surrounding vessels were counted for all tumors.The Chick Chorioallantoic Membrane (CAM) model was performed as described before ,26,27. Tt-test was performed and significance was assumed for p < 0.05.Statistics were performed using GraphPad by Dotmatics. A In conclusion, extracellular citrate uptake via the pmCiC is a potential novel therapeutic target in MCC tumors that highly expresses this critical citrate transporter, especially for cancers that tend to be more aggressive or metastatic. The exact mechanisms that play a role in this effect will require further investigation.


Aiming to reduce the gap between in vitro and in vivo environment, a complex culture medium, Plasmax, was introduced recently, which includes nutrients and metabolites with concentrations normally found in human plasma. Herein, to study the influence of this medium on cellular behaviors, we utilized Plasmax to cultivate two cancer cell lines, including one breast cancer cell line, MDA-MB-231BR, and one brain cancer cell line, CRL-1620. Cancer cells were harvested and prepared for transcriptomics and proteomics analyses to assess the discrepancies caused by the different nutritional environments of Plasmax and two commercial media: DMEM, and EMEM. Total RNAs of cells were extracted using mammalian total RNA extract kits and analyzed by next-generation RNA sequencing; proteomics analyses were performed using LC-MS/MS. Gene oncology and pathway analysis were employed to study the affected functions. The cellular invasion and cell death were inhibited in MDA-MB-231BR cell line when cultured in Plasmax compared to DMEM and EMEM, whereas the invasion, migration and protein synthesis of CRL-1620 cell line were activated in Plasmax in relative to both commercial media. The expression changes of some proteins were more significant compared to their corresponding transcripts, indicating that Plasmax has more influence upon regulatory processes of proteins after translation. This work provides complementary information to the original study of Plasmax, aiming to facilitate the selection of appropriate media for in vitro cancer cell studies. Due to the different nutrient compositions and concentrations, the culturing environment of media can greatly affect the cell metabolism ,2,3, andRecently, more attentions have been drawn to the optimization of the nutrient compositions and concentrations of culturing media for cancer cell line cultivation ,13,14,15In this study, we made comparisons between Plasmax and two commercially available media, DMEM and EMEM by investigating the expression changes of transcriptomes and proteomes of cancer cell lines to obtain more comprehensive understandings about how this novel medium can affect the cellular functions. A breast cancer cell line (MDA-MB-231BR), and a brain cancer cell line (CRL-1620) were subject to cultivation in the abovementioned three different culturing media and cell harvest. Total RNAs and proteins were extracted and prepared to evaluate the gene and protein expressions, respectively. Unsupervised principal component analysis (PCA) was applied to assess the expression differences within two cancer cell lines cultured by three media. Gene ontology analysis and protein function annotation were performed utilizing Ingenuity pathway analysis software (QIAGEN IPA) to study the altered cellular functions. Significant up- or down-regulations of proteins that are associated with cell movement, cell death and survival, and protein synthesis were observed.Breast cancer cell line MDA-MB-231BR (231BR) was generously provided by Dr. Paul Lockman . Brain cancer cell line CRL-1620 (CRL), DMEM, EMEM, and Fetal Bovine Serum (FBS) were acquired from American Type Culture Collection . Corning Trypsin EDTA 1× (25% Trypsin/2.21 mM EDTA), MS-grade formic acid (FA), HPLC grade water, methanol, and acetonitrile (ACN) were obtained from Fisher Scientific . Mammalian total RNA extraction kit, sodium hydroxide beads, Dithiothreitol (DTT), iodoacetamide (IAA), ammonium bicarbonate (ABC) and sodium deoxycholate (SDC) were purchased from Sigma-Aldrich . NEBNext rRNA depletion fit (human/mouse/rat), and NEBNext Ultra II Directional RNA library prep kit were obtained from New England Biolabs . Trypsin/Lys-C mix, mass-spectrometry-grade, was purchased from Promega .Plasmax medium was prepared based on the formulation  and follTo keep the stock solutions from being contaminated, the mixing process was performed in a strictly aseptic environment. A bottle of 500 mL Plasmax medium was prepared according to the following steps: First, 5mL of stock solution #1 and #2 were added into 482 mL of EBSS, followed by 0.5 mL of solutions #3 and #8. Then, 1mL of solutions #4 and 0.05 mL of #6 were added. Next, 5 mL of BME-Vitamin, 1.625 mL of L-Glutamine and 12.5 mL of fetal bovine serum (FBS) were also added. After adding all stock solutions and other required compositions, the mixture was shaken for 10 min to help the medium mix evenly. Then, the mixture was pushed through a Nalgene rapid-flow filter  with a sterilizing-grade 0.2 μm SFCA membrane installed to remove any bacteria that potentially involved in the medium solution. Finally, the well-prepared 500 mL Plasmax medium was stored in 4 °C for future use.2 flasks. Cell culture was incubated at 37 °C for 4–8 days and fed every 2–4 days. When the cell confluence reached 90%, cells were washed with a 10mL aliquot of PBS twice and detached by 2.2 mL trypsin-EDTA solution. After incubating at 37 °C for 5 min, 5 mL of fresh medium was added to the cell solution to quench trypsin. The cell suspension solution was then moved into three 175 cm2 flasks for biological triplicate and incubated at 37 °C for about 7 days, until it reached 80% of cell confluence. Cells were washed and detached from the flasks using the abovementioned method. After adding 10 mL of fresh medium, cells were harvested by centrifuging at 500× g for 5 min. Cell pellets were collected and washed twice with PBS to get rid of the extra medium. Part of the fresh cells were taken for RNA extraction. The rest of cell samples were stored in −20 °C for future proteomics analyses.The cancer cell lines were cultured and harvested according to the previously reported procedures ,19. Two Cancer cells were first washed with PBS solution and total RNAs were extracted from the cells using a Mammalian Total RNA Extraction Kit following the procedure provided by the vendor. For each cell line, three biological replicates were prepared. After the extraction, a 5-μL aliquot of total RNA solution was taken to determine the concentration using a Nanodrop 1000 spectrophotometer . Then, RNA quality was determined using RNA Screen Tape . Ribosomal RNA depletion was achieved using a NEB Next rRNA Depletion Kit (Human/Mouse/Rat). RNA fragmentation, double stranded cDNA, and adaptor ligation was generated using NEBNext Ultra II Directional RNA Library Prep following the manufacturer’s procedures. PCR enriched libraries were quantified by Qubit and equimolar indexed libraries were pooled. Pooled libraries were quantitatively checked using software Tapestation 2200  and quantified using Qubit. The libraries were then diluted to 200 pM and spiked with 2% phiX libraries (Illumina control). The transcriptome sequencing was performed on the barcoded stranded RNA-Seq libraries using Illumina NovaSeq 6000 S1 flow cell , paired-end reads (2 × 100 bp).g for 10 min. The supernatant, which contained the extracted cell proteins, was collected. Extracted protein from 231BR and CRL cell lines were then treated by tryptic digestion [The protein extraction was accomplished by following previously reported procedures ,21,22. Tigestion ,25. BrieBased on the protein concentration, the remaining samples were first diluted to make a 0.5% concentration of SDC, then reduced by adding 200 mM dithiothreitol (DTT) and incubated at 60 °C for 45 min. Then, the reduced proteins were alkylated by adding 200 mM iodoacetamide (IAA) followed by incubation at 37.5 °C for 45 min in dark. The alkylation reaction was quenched by adding 200 mM DTT again and incubated at 37.5 °C for 30 min. The pH value of each sample was checked before the addition of enzyme to make sure the high efficiency of enzymatic digestion. The protease trypsin/Lys C ,27, was t test was applied to search for significant changes of protein abundances (p < 0.05), followed by Benjamini-Hochberg (BH) procedure for the multiple testing correction. Data sets of significant proteins from both cell lines were uploaded to Ingenuity Pathway Analysis  for functional annotation and pathway analysis to investigate the cellular function discrepancies correlated with the altered protein expressions.The raw data files generated by LC-MS/MS were processed using software MaxQuant v2.0.2.0  . ProteinProteomics analyses were performed using a Dionex UltiMate 3000 nano LC system  coupled with an LTQ Orbitrap Velos mass spectrometer  through a nano-ESI source. A C18 Acclaim PepMap 100 trapping column  was used to remove salt and impurities. Peptide was separated by a C18 Acclaim PepMap RSLC column . The column compartment temperature was set at 29.5 °C. Mobile phase A contained 2% of ACN and 0.1% of FA, while mobile phase B had 0.1% FA in ACN. A 120 min elution method was utilized for the separation of peptides. The gradient of mobile phase B started at 5% over the first 10 min, then increased to 20% in 55 min, 20% to 30% in 25 min, 30% to 50% in 20min, then ramped up to 80% in 1 min and stayed at 80% for 4 min, then dropped back to 5% in 1 min and stayed for 4 min. Two MS scan events were applied for data acquisition, including a full MS scan with the m/z range of 400–2000 at a resolution of 60,000, and a CID MS/MS scan using normalized collision energy (CE) of 35% and a 10 ms activation time. A data dependent acquisition (DDA) method was employed, which selected the top eight most intense ions from the full scan for the MS/MS analysis.www.serummetabolome.ca, accessed on 11 January 2019) [The concentrations of the nutrients and metabolites of the Plasmax media was based on the freely available resource (ry 2019) . Supportp-values between Plasmax and commercial media were calculated using student’s t test. For 231BR cell lines cultivated in Plasmax, a total of 536 and 265 genes were found as statistically significant (p < 0.05) when compared to this cell line cultivated in DMEM and EMEM, respectively. On the other hand, the CRL cell lines cultivated by Plasmax included 349 and 712 significant genes in relative to DMEM and EMEM, respectively. The Venn diagrams in Next generation RNA sequencing was performed to investigate the alterations of gene expressions among the cells cultured in the different media. Overall, 19973 genes from 231BR, and 20465 from CRL were identified . Based oIn gene ontology analysis, differently expressed transcriptomes were clustered according to their molecular and cellular functions ,32. Compp-values of statistically significant transcriptomes were uploaded to IPA. IPA will search against its database and predict the corresponding upstream regulators and the influence on downstream processes based on the given data. In breast cancer cell line 231BR, as shown by In order to gain more understandings about the impacts on cellular functions due to different transcriptome expressions in Plasmax, the pathway analysis was performed using QIAGEN IPA. The two commercial media were employed as baseline, and the transcriptomics results acquired from cell lines cultivated in Plasmax were compared with the counterparts in DMEM and EMEM, respectively. The logarithm of fold changes and 2+ and calmodulin can activate calcium/calmodulin dependent protein kinase (CAMK) and lead to phosphorylation [For the brain cancer cell line CRL, the transcriptomics discrepancies between Plasmax and DMEM resulted in the inhibition of the calcium signaling pathway. As shown in rylation , then acrylation . It has rylation , suggestrylation . On the rylation .t test was then utilized to search for significant changes of protein abundances between Plasmax and commercial media (p < 0.05). In 231BR cell lines , which also suggests that Plasmax might have more impacts on the expressions of proteomes than transcriptomes during the cell line cultivation.To investigate the different expressions of these proteins from transcriptomics level and the correlation between the proteome and the transcriptome, the By using EMEM as the baseline, the inhibition of integrin signaling pathway of 231BR cell line in Plasmax were also observed. As shown by The PAK2 that mentioned above in integrin signaling pathway is also involved in PAK signaling pathway , which iBased on the significant changes of protein expressions, IPA also predicted the upstream regulators and downstream functions. As depicted by In contrary to the 231BR cell line, the cell movement capability, such as cell invasion and migration of brain cancer cell line CRL were more activated when cultured in Plasmax than both DMEM and EMEM. Additionally, the functions related to protein expressions and synthesis were also stimulated by Plasmax. The abovementioned function alterations are associated with the activation of unfolded protein response (UPR) signaling pathway as shown in UPR is an adaptive mechanism in eukaryotic cells to response to the endoplasmic reticulum (ER) stress of misfolded proteins . The prop < 0.01) of proteins involved in the downstream signaling pathway, including heat shock 70 kDa protein 9 (HSPA9) and calnexin (CANX) were also observed in Plasmax. However, similar as discussed in 231BR cell line, the transcripts of HSPA9 and CANX did not present any significant expression changes, (p > 0.9) suggesting that the expressions of these downstream proteins are not only regulated by their transcripts but also controlled by other regulatory processed after transcriptions.In our work, the chaperon GRP78 of CRL cell line and its corresponding transcript HSPA5 were up-regulated by 41% and 28%, respectively, when cultivated in Plasmax than in DMEM. (inset table of Another important finding of CRL cell line is the overexpression of 14-3-3 family proteins. By binding to the phosphorylated serine or threonine residues of the target proteins , 14-3-3 In this study, we investigated and compared the physiological cell culturing media, Plasmax with two commercially available media DMEM and EMEM through transcriptomics and proteomics. Significant differences of gene and protein expressions were observed in two cancer cell lines cultured by these three media. The gene oncology and pathway analyses of significant transcriptomes suggest alterations in cellular functions. The cell proliferation and invasion of 231BR were inhibited by Plasmax compared to DMEM. However, these functions were more activated than in EMEM. For CRL, the calcium signaling pathway suggested inhibition of cell growth and development in Plasmax in relative to DMEM, while cell cycle was hindered compared to EMEM. On the other hand, the GO analyses of significant proteins indicate that cellular movement, and cell death and survival were the top affected cellular functions in both breast cancer and brain cancer cell lines. According to the expression changes of proteins in 231BR cell line, it also exhibits less cellular invasiveness in Plasmax compared to both commercial media. However, for CRL cell line, its invasion and migration were predicted to be enhanced in Plasmax. The protein synthesis in CRL was another promoted function by Plasmax in relative to DMEM and EMEM. Moreover, the cell death of both cancer cell lines was inhibited when cultivated in Plasmax than both commercial media. The pathway analysis of proteins was utilized to further assess the cellular function alterations introduced by different media. Compared to the commercial media, breast cancer cell line 231BR cultivated in Plasmax exhibited the inhibition of integrin signaling, and PAK signaling pathways, leading to the decrease in cell invasion and cell death, while the activation of unfolded protein response signaling and 14-3-3 mediated signaling pathways in brain cancer cell line CRL are associated with enhanced protein synthesis, cellular migration and invasion.The comparisons between the protein expressions and their corresponding transcriptomes involved in the inhibited or activated pathways suggest that Plasmax has more impacts on these protein expressions, since the RPKM changes of the genes were not as significant as the proteins. In addition, both of transcriptomics and proteomics analyses of 231BR cell line indicated that the invasiveness was inhibited by Plasmax in relative to DMEM. However, the transcriptomics and proteomics analyses suggested opposite results when compared to EMEM. Additionally, the pathway analyses of significant genes and proteins in CRL cell line led to different cellular functions that were affected. The inconsistency between transcriptomics and proteomics indicates that Plasmax might affect the protein expressions after the translations, thus resulting in unique function alterations in proteomics analyses. It can be expected due to the complicated regulation between proteins and their transcripts, with different level of enzymes involved. Overall, these variances of gene and protein expressions indicate the potential influence from the nutrient environment of Plasmax and result in cell behavior changes in different types of cancer cell lines. Our investigation of transcriptomics and proteomics provided complementary information to the original study which focused on the cell proliferation and metabolomics. Researchers may find more clues about media selection for in vitro cell-based transcriptomics or proteomics in future studies.


G12C protein have been developed and demonstrate clinical response rates of 37-43%. Importantly, these agents fail to generate durable therapeutic responses with median progression-free survival of ~6.5 months.The KRAS(G12C) mutation is the most common genetic mutation in North American lung adenocarcinoma patients. Recently, direct inhibitors of the KRASG12C-driven lung cancer cell lines. The co-occurring NRASQ61L mutation in KRASG12C-positive LLC cells was deleted and the KRASG12V allele in CMT167 cells was edited to KRASG12C with CRISPR/Cas9 methods. Also, a novel murine KRASG12C line, mKRC.1, was established from a tumor generated in a genetically-engineered mouse model.To provide models for further preclinical improvement of these inhibitors, we generated three novel murine KRASin vitro sensitivities to KRASG12C inhibitors , but distinct in vivo responses to MRTX-849 ranging from progressive growth with orthotopic LLC-NRAS KO tumors to modest shrinkage with mKRC.1 tumors. All three cell lines exhibited synergistic in vitro growth inhibition with combinations of MRTX-1257 and the SHP2/PTPN11 inhibitor, RMC-4550. Moreover, treatment with a MRTX-849/RMC-4550 combination yielded transient tumor shrinkage in orthotopic LLC-NRAS KO tumors propagated in syngeneic mice and durable shrinkage of mKRC.1 tumors. Notably, single-agent MRTX-849 activity in mKRC.1 tumors and the combination response in LLC-NRAS KO tumors was lost when the experiments were performed in athymic nu/nu mice, supporting a growing literature demonstrating a role for adaptive immunity in the response to this class of drugs.The three lines exhibit similar G12C mutant lung cancer should prove valuable for identifying improved therapeutic combination strategies with KRASG12C inhibitors.These new models of murine KRAS G12C inhibitor sotorasib (AMG-510) achieved an objective response rate of 37.1% and the median duration of response was 11.1 months with a comparatively short progression free survival of 6.8 months  state. Distinct from other KRAS codon 12 alleles, KRASG12C retains intrinsic GTP hydrolysis capacity  supplemented with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin  at 37°C in a humidified 5% CO67 cells  and Lewiem1LdowKras/J mice  were crossed with B6.129P2-tm1BrnTrp53/J mice (Jackson Labs stock #008462). F1 hybrid mice were genotyped for KRAS and p53 status using PCR on genomic DNA and the following primer pairs: KRASG12C, LSL-Kras Common 5’-TCCAATTCAGTGACTACAGATG-3’, LSL-Kras Mutant 5’-CTAGCCACCATGGCTTGAGT-3’, LSL-Kras Wildtype 5’-ATGTCTTTCCCCAGCACAGT-3’); Trp53, FWD 5’-GGTTAAACCCAGCTTGACCA-3’, REV-5’-GGAGGCAGAGACAGTTGGAG-3’). All PCR reactions employed PCRBIO VeriFi™ mix  according to manufacturer’s protocol with 60°C annealing temperature. Mice heterozygous for KRASG12C and varying p53 backgrounds  were inoculated with Ad-Cre intratracheally and submitted to microCT imaging once monthly until tumor burden was detected. Tumors were harvested, minced, plated on tissue culture plastic and cultured until the stable cell line, mKRC.1, was obtained.B6;129S4-2 plate and cultured for 16 hrs. The NRAS targeting crRNA, ATTO550-labelled tracrRNA and recombinant Cas9 were assembled into ribonucleoprotein (RNP) particles according to IDT manufacturer’s protocol. Assembled RNPs were packaged using Lipofectamine RNAiMAX transfection reagent (ThermoFischer Scientific) according to manufacturer’s instructions, and transfection mixture was added to LLC cells. After a 24-hour incubation, cells were trypsinized, rinsed with 5 mL of PBS, and resuspended into a single-cell suspension in 1 mL of PBS. Single cells were flow sorted into 96-well plates using the MoFlo XDP100 cell sorter for ATTO550 positivity (Beckman Coulter) . For proper compensation of flow cytometry channels, unstained cell lines were used. Single cell clones were expanded, genomic DNA collected, and assessed for NRAS perturbations via PCR and Sanger Sequencing. Two independent clones were identified and indicated as LLC 23 NRAS KO and LLC 46 NRAS KO.A crRNA was designed targeting exon 5 of genomic mouse NRAS using IDT  online software (5’-CACGAACTGGCCAAGAGTTA-3’). LLC cells  were plated in a 35 mmG12C cell lines were generated by the same protocol used for generating LLC NRAS knockout cell lines with the addition of two steps. 1. Prior to transfection, CMT167 cells were incubated with 30 μM HDR Enhancer (IDT) for 1 hour. 2. ssODN’s were added to the transfection mixture and co-transfected into cells along with assembled RNP’s. Single cell subclones from flow sorting were expanded and analyzed for endogenous KRASG12C mutations.A crRNA targeting exon 1 of genomic mouse KRAS (5’-GACTGAGTATAAACTTGTGG-3’) using IDT online software was designed in addition to single stranded donor oligonucleotides (ssODN) homology-directed repair templates for both the positive and negative strands, the sequence for which is: 5’-ATGACTGAGTATAAACTTGTCGTCGTTGGAGCTTGCGGCGTAGGCAAGAGCGCCTTGACGATACAGCTAATTCAGA-3’. CMT167 KRASRNA was submitted to the University of Colorado Sequencing Core where library preparations were generated, and RNA was sequenced on the NovaSeq 6000 to generate 2 x 151 reads. Fastq files were quality checked with FastQC, illumina adapters trimmed with bbduk, and mapped to the mouse mm10 genome with STAR aligner. Counts were generated by STAR’s internal counter and reads were normalized to counts per million (CPM) using the edgeR R package. Differential expression was calculated using the limma R package and the voom function. Heatmaps were generated in Prism 9 . Integrated Genomics Viewer analysis was completed by stripping section of sequence reads from RNAseq dataset and viewing in IGV software .Fastq files were quality checked with FastQC, illumina adapters trimmed with bbduk, and paired reads were mapped to the UCSC mm10 BWA genome with samtools bwa-mem. Output sam files were converted to bam format, sorted, and duplicates were marked with the picard tool, MarkDuplicates. Samtools “mpileup” and bcftools “call” multiallelic caller were used to call variants and generate variant call files (VCF). To identify variants unique to each cell line, bcftools “isec” was used on each cell lines VCF files filtered with bcftools “view” excluding “G/T = 0/0” and including variants with at least a read depth of 10 and a quality score of 20 compared to unfiltered parental VCF files. Variants private to each cell line (001.vcf) were then annotated with ANNOVAR mm10db including refGene, cytoBand, and genomicSuperDups. VCFs were filtered excluding identified “intronic”, and “intergenic” annotated variants under the “Func.refGene tab in addition to “synonymous”, “.”, and “other” under the “ExonicFunc.refGene” tab. Variants were confirmed by interrogating specific regions from the BAM files and uploading those reads to the integrated genomics viewer (IGV). A variant was considered detected reliably if the variant was present in > 10% of the total read depth at the variant position. All code and the parameters used are not included in this report but can be provided upon request.Cells were plated at 100 cells per well in 100 μL in 96-well tissue culture plates and allowed to attach for 24 hrs. Inhibitors were added at various doses as 2X concentrates in 100 μL. After incubation for 7-10 days, cell number per well was assessed using a CyQUANT Direct Cell Proliferation Assay (Invitrogen) according to the manufacturer’s instructions. The data are means and SEM (n=3) and presented as percent of the control values measured in DMSO-treated wells for each cell line.Conditioned media was collected from treated and untreated murine lung cancer cell lines. Chemokine levels were measured using the Invitrogen ELISA Kit  following manufacturer’s instructions. Absorbance was measured at 450 nm. The measured concentration in each sample was normalized to the total cellular protein per dish and the data are presented as pg/μg protein.nu/nu mice were purchased from Envigo . For tumor cell inoculation, parental LLC, LLC 46 NRAS KO, LLC 23 NRAS KO and mKRC.1 cells were grown to 75% confluence, harvested, and resuspended in sterilized phosphate buffered saline. Cells were counted and resuspended to a final concentration of 125,000 cells per 40uL injection. Cell suspensions were directly injected into the left lung of mice through the ribcage. Mice were randomized into groups to receive diluent control, RMC-4550 , MRTX-849 , or the combination by daily oral gavage until primary experimental endpoints were met. Treatment was initiated following confirmation that at least 75% of mice had calculatable primary left lung tumors via microCT imaging . MicroCT imaging was conducted weekly and ITK-SNAP software  and eight-week-old software  was used50 values, the dose-response data were submitted to the “log(inhibitor) vs. normalized response” function with the Prism 9 software. An unpaired Student’s t test (two-tail) was used to determine statistical significance, unless otherwise noted. The P values are denoted by *(P < 0.05), **(P < 0.01), ***(P < 0.001), and ****(P < 0.0001) and were corrected for multiple comparisons (Dunnett). Drug synergy with combinations of KRAS-G12C inhibitors and SHP2 inhibitors was determined with Combenefit, a free software tool for visualization, analysis and quantification of drug combination effects. Data from drug combination assays was processed using the HSA synergy model.Statistical Analysis: Prism 9  was used to perform specific statistical analyses noted in the figure legends. Data are presented as the mean and standard error of the mean (SEM) as indicated. To calculate ICG12C-driven LUAD cell lines to permit investigation of KRASG12C inhibitor responses in immune-competent hosts.The literature supports a role for innate and adaptive immunity in the overall tumoral responses to oncogene-specific inhibitors , 27, 28.G12C mutation  targeting exon 5 of the murine NRAS gene was introduced into parental LLC as a complex with Cas9 and single-cell transfectants were isolated and screened for functional NRAS knockout. Genetic NRAS knockout (KO) in 2 independent LLC clones (LLC 23 NRAS KO and LLC 46 NRAS KO) was validated by RNAseq showing a large deletion near the gRNA targeting region of exon 5 of NRAS (Lewis lung carcinoma (LLC) are derived from a spontaneous lung tumor that arose in a C57BL/6 mouse  and has mutation . Variantmutation  verifiedG12V mutation. CMT167 cells express wild-type Trp53. CRISPR-Cas9-mediated, homology-directed repair was deployed to edit the endogenous G12V mutation to a G12C mutation. Ribonucleoprotein (RNP) particles containing Cas9, a single-stranded oligonucleotide DNA template and a gRNA targeting a sequence within exon 2 of murine KRAS were prepared and transfected into parental CMT cells. Following single cell flow sorting, clones were submitted to functional screening assessed for acquired sensitivity to KRASG12C inhibitors. Putative positive clones that successfully underwent homology-directed repair to contain the KRASG12C mutation were submitted to RNAseq and expression of the G12C allele was verified. RNA sequencing and IGV analysis confirmed an indel in one allele of murine KRAS and a successful recombination containing both KRASG12C as well as the predicted wobble position switches engineered to prevent Cas9 re-cutting of successfully recombined KRASG12C gene  and crossed with Trp53fl/fl mice . The resulting KRASG12C/+; Trp53fl/fl mice were identified by genotyping and submitted to intratracheal administration of Ad-Cre as previously described . Knockout of NRAS increased the sensitivity of LLC NRAS KO cell lines to both KRASG12C inhibitors  compared to parental LLC cells  and indicate that combination therapy with MRTX-849 will be required to achieve clinically significant anti-tumor responses. In this regard, the protein tyrosine phosphatase, PTPN11/SHP2, has emerged as a promising upstream target as it functions to dephosphorylate multiple proteins leading to sustained KRAS GTP cycling and activation . The results revealed a modest reduction in the rate of growth by single agent KRAStivation , 40, 41,tivation  and T cetivation , 15. Bottivation –17.G12C inhibition, we plated LLC 46 NRAS KO, CMT-KRAS-G12C.54 and mKRC.1 cell lines in 96-well plates and assessed cell growth in response to a range of concentrations of MRTX-1257 and RMC-4550 alone and in combination. Analysis of the resulting data  or rapid progression after initial shrinkage (mKRC.1) to MRTX-849 as a single agent. Thus, these new murine KRASG12C-driven lung cancer cell lines propagated as orthotopic tumors reflect treatment responses in KRASG12C-positive patients treated with single agent KRASG12C inhibitors  form  form . Unlike ant form . This re and SOS .https://www.ncbi.nlm.nih.gov/geo/,GSE207098.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by University of Colorado Institutional Animal Care and Use Committee.Conception and design: DS, TH, EK, RN, LH. Development of methodology: DS, TH, AL, RN, LH. Acquisition of data: DS, TH, LH. Analysis and interpretation of data: DS, TH, LH. Writing, review, and/or revision of the manuscript: DS, TH, EK, RN, LH. All authors contributed to the article and approved the submitted version.