Intratumoral heterogeneity of the therapeutical response to gemcitabine and metformin

Abstract

Cancer heterogeneity and microenvironmental aspects within a tumor are considered key factors influencing resistance of carcinoma cells to distinct chemotherapeutical agents. We evaluated a high concentration of metformin in combination with gemcitabine on a syngeneic orthotopic mouse model using 6606PDA cells. We observed reduced tumor size and reduced cancer cell proliferation after three weeks of chemotherapy with either compound and noticed an additive effect between gemcitabine and metformin on tumor weight. Interestingly, distinct areas of the carcinoma responded differently to either compound. Metformin inhibited the proliferation of cancer cells close to the desmoplastic reaction, whereas gemcitabine inhibited the proliferation of cancer cells mainly 360–570 μm distant to the desmoplastic reaction. Indeed, co-culture of pancreatic stellate cells with 6606PDA, 7265PDA or MIA PaCa-2 cells increased gemcitabine resistance. Metformin resistance, however, was increased by high glucose concentration in the medium. Other factors such as hypoxia or the pH of the medium had no influence on gemcitabine or metformin induced inhibition of cancer cell proliferation. These data demonstrate a spatial heterogeneity in drug resistance within pancreatic adenocarcinomas and that microenvironmental aspects such as supply of glucose and the presence of pancreatic stellate cells regulate cancer cell sensitivity towards metformin or gemcitabine.

Metformin Inhibits Gemcitabine Induced Apoptosis in Pancreatic Cancer Cell Lines

“…Surprisingly, few other publications support this point of view. Many publications describe that metformin plus gemcitabine treatment increases cell death in various pancreatic cancer cell lines when compared to the monotherapies 11 , 12 , 13 or describe other benefits of combining metformin with gemcitabine 10 , 14 - 17 . Only very few publications reported that metformin inhibits for example cisplatin induced apoptosis of cancer cell lines 18 or apoptosis in primary cells 19 .…”

“…For evaluating the influence of hypoxia or normoxia on cell lines, the cells (covering about 15% of the well surface) were incubated with DMEM high glucose medium or DMEM high glucose medium containing therapeutic agents for 72 hours either in an Innova CO-48 incubator (New Brunswick Scientific Co, Edison Edison, USA) under 1 % oxygen supply or in an incubator with normoxic conditions. For evaluating the influence of pH-value on cell lines, the cells were incubated for another 48 hours with DMEM high glucose medium adjusted to pH 6.8 or pH 7.8 with 1M HCl or 1M NaOH as previously described 10 . The medium with pH 6.8 was also supplemented with 10 mM lactate (Sigma-Aldrich St Louis, MO, USA) in order to mimic lactate production in the carcinoma.…”

“…On the following day the cells covered about 30% of the well surface and were incubated for another 24 or 48 hours with control medium or medium containing therapeutic agents (as described above). After separating the cell lysates on SDS polyacryl gels the proteins were transferred to a polyvinyldifluoride membrane (Immobilon-P; Millipore, Eschborn, Germany) as described previously 10 . The membranes were blocked with 2.5 % (wt/vol.)…”

“…Data presentations and statistics using a Mann-Whitney rank-sum test followed by the Bonferroni correction were described previously 10 . Differences with P ≤ 0.05 or P ≤ 0.08, divided by the number of meaningful comparisons, were considered to be significant or to indicate a tendency.…”

The role of stromal cancer-associated fibroblasts in pancreatic cancer

“…Little is known though about the character and function of stroma within PDAC metastases [43]. Zechner et al did find that, within primary tumors treated with a combination of metformin and gemcitabine, metformin better inhibited cancer cells near the desmoplastic stroma, whereas gemcitabine inhibited proliferation in cells distant to the stroma [48]. Hesler et al showed that CAFs serve as sources of CYR61 in co-culture models and induce chemoresistance by downregulating nucleoside transporters that mediate cellular uptake of gemcitabine [49].…”