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Patient-derived xenografts (PDX) are being increasingly utilized in preclinical oncologic research. Maintaining large colonies of early generation tumor-bearing mice is impractical and cost-prohibitive. Optimal methods for efficient long-term cryopreservation and subsequent reanimation of PDX tumors are critical to any viable PDX program. We sought to compare the performance of “Standard” and “Specialized” cryoprotectant media on various cryopreservation and reanimation outcomes in PDX tumors. Standard (10% DMSO media) and Specialized (Cryostor®) media were compared between overall and matched PDX tumors. Primary outcome was reanimation engraftment efficiency (REE). Secondary outcomes included time to tumor formation (TTF), time to harvest (TTH), and potential loss of unique PDX lines. Overall 57 unique PDX tumors underwent 484 reanimation engraftment attempts after previous cryopreservation. There were 10 unique PDX tumors cryopreserved with Standard (71 attempts), 40 with Specialized (272 attempts), and 7 with both (141 attempts). Median frozen time of reanimated tumors was 29 weeks (max. 177). Tumor pathology, original primary PDX growth rates, frozen storage times, and number of implantations per PDX model were similar between cryoprotectant groups. Specialized media resulted in superior REE (overall: 82 vs. 39%, p < 0.0001; matched: 97 vs. 36%, p < 0.0001; >52 weeks cryostorage: 59 vs. 9%, p < 0.0001), shorter TTF (overall 24 vs. 54 days, p = 0.0051; matched 18 vs. 53 days, p = 0.0013) and shorter TTH (overall: 64 vs. 89 days, p = 0.009; matched: 47 vs. 88 days, p = 0.0005) compared to Standard. Specialized media demonstrated improved REE with extended duration cryostorage (p = 0.048) compared to Standard. Potential loss of unique PDX lines was lower with Specialized media (9 vs. 35%, p = 0.017). In conclusion, cryopreservation with a specialized cryoprotectant appears superior to traditional laboratory-based media and can be performed with reliable reanimation even after extended cryostorage.

Lumican is secreted by pancreatic stellate cells and inhibits cancer progression. Extracellular lumican inhibits cancer cell replication and restrains growth of early-stage pancreatic adenocarcinoma (PDAC) such that patients with tumors containing stromal lumican experience a three-fold longer survival after treatment. In the present study, patient tumor tissues, ex-vivo cultures of patient-derived xenografts (PDX), PDAC stellate and tumor cells were used to investigate whether hypoxia (1% O2) within the tumor microenvironment influences stromal lumican expression and secretion. We observed that hypoxia significantly reduced lumican expression and secretion from pancreatic stellate cells, but not cancer cells. Although hypoxia enhanced lactate dehydrogenase A (LDHA) expression and lactate secretion from all cells, neither hypoxia-induced nor exogenous lactate influenced lumican expression. Autophagy was induced by hypoxia in ex vivo cultures of PDX and pancreatic stellate cells, but not cancer cells cultured in 2D. Autophagic flux inhibitors, bafilomycin A1, chloroquine diphosphate salt, and ammonium chloride prevented hypoxia-mediated reduction in lumican expression in stellate cells. Furthermore, inhibition of AMP-regulated protein kinase (AMPK) phosphorylation or hypoxia-inducible factor (HIF)-1α expression within hypoxic stellate cells restored lumican expression levels. Hypoxia did not affect lumican mRNA expression, indicating that hypoxia-induced reduction of lumican occurs post-transcriptionally; in addition, AMPK inhibition prevented hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway, a key contributor to protein synthesis. Taken together, these findings demonstrate that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells.

Although data suggests little hope for survival when patients present with metastatic pancreatic cancer, recent advances in systemic therapy offer the possibility for dramatic tumor responses like those observed in other disease sites. Here, we present the case of a 50-year-old woman who presented with adenocarcinoma of the pancreas with two liver metastases and a CA 19-9 level of 1,659 U/mL. The patient received FOLFIRINOX (leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin) with a dramatic reduction in CA 19-9 level to 23.9 U/mL, and complete regression of both liver metastases. The patient then received capecitabine with the maintenance of a normal CA19-9 over the next 12 months. With no evidence of distant disease, concurrent systemic and local therapy with capecitabine-based chemoradiation (CapeXRT) was performed followed by observation for eight months with normal CA 19-9 readings. A mild increase in CA 19-9 (143 U/mL) prompted a restaging demonstrating an active primary tumor but no distant disease. Therefore, a pancreaticoduodenectomy (PD or Whipple) was performed rendering this patient free of detectable cancer. Our team has developed an ex-vivo chemosensitivity assay in which the tumor tissue from an individual patient can be rapidly examined for sensitivity to available systemic therapy treatment strategies. We tested this patient's tumor for its sensitivity to gemcitabine (Gem) versus a combination of 5-fluorouracil, irinotecan, and oxaliplatin (FIRINOX). Remarkably, our assay confirmed a profound sensitivity of this patient's tumor to the agents she had received.

Lumican is secreted by pancreatic stellate cells and inhibits cancer progression. Extracellular lumican inhibits cancer cell replication and restrains growth of early-stage pancreatic adenocarcinoma (PDAC) such that patients with tumors containing stromal lumican experience a three-fold longer survival after treatment. In the present study, patient tumor tissues, ex-vivo cultures of patient-derived xenografts (PDX), PDAC stellate and tumor cells were used to investigate whether hypoxia (1% O2) within the tumor microenvironment influences stromal lumican expression and secretion. We observed that hypoxia significantly reduced lumican expression and secretion from pancreatic stellate cells, but not cancer cells. Although hypoxia enhanced lactate dehydrogenase A (LDHA) expression and lactate secretion from all cells, neither hypoxia-induced nor exogenous lactate influenced lumican expression. Autophagy was induced by hypoxia in ex vivo cultures of PDX and pancreatic stellate cells, but not cancer cells cultured in 2D. Autophagic flux inhibitors, bafilomycin A1, chloroquine diphosphate salt, and ammonium chloride prevented hypoxia-mediated reduction in lumican expression in stellate cells. Furthermore, inhibition of AMP-regulated protein kinase (AMPK) phosphorylation or hypoxia-inducible factor (HIF)-1α expression within hypoxic stellate cells restored lumican expression levels. Hypoxia did not affect lumican mRNA expression, indicating that hypoxia-induced reduction of lumican occurs post-transcriptionally; in addition, AMPK inhibition prevented hypoxia-reduced phosphorylation of the mTOR/p70S6K/4EBP signaling pathway, a key contributor to protein synthesis. Taken together, these findings demonstrate that hypoxia reduces stromal lumican in PDAC through autophagy-mediated degradation and reduction in protein synthesis within pancreatic cancer stellate cells.