Fig 2: Aspirin, oseltamivir phosphate, and gemcitabine modify the expression of hypoxia markers in pancreatic cancer tumors. (A) Expression of HIF-1α (B) HIF-2α, (C) CAIX, and (D) pimonidazole in pancreatic tumor tissue following mice indicated treatments with ASA/OP in combination with GEM. For pimonidazole tumor tissues, mice received an IP injection of 15 mg of pimonidazole/mouse 1 h prior to euthanasia. Tissue images were captured using the Nikon Eclipse EI R STG HNDL TRINOC microscope equipped with digital sight 1000 microscope camera using the 40× objective. Scale bars represent 200 μm. Relative pixel density of (E) HIF-1α, (F) HIF-2α, (G) CAIX, and (H) pimonidazole. Relative density was reported as mean ± SEM of 3–5 slides per treatment. The one-way ANOVA Fisher test was used for comparison tests at 95% confidence intervals. The indicated asterisks represent the statistical significance compared to the untreated CTRL or otherwise shown. Abbreviations: CAIX—carbonic anhydrase 9; ASA—aspirin; OP—oseltamivir phosphate; GEM—gemcitabine; CTRL—control; SEM—standard error of the mean.

Fig 3: ASA and OP, in combination with GEM, inhibit the development of EMT markers in pancreatic tumors. (A) Expression of N-cadherin, (B) E-cadherin, (C) EpCAM, and (D) vimentin on pancreatic tumor tissue following treatment with ASA/OP/GEM. Tissue images were captured using the Nikon Eclipse EI R STG HNDL TRINOC microscope equipped with digital sight 1000 microscope camera using the 40× objective. Scale bars represent 200 μm. Relative density of (E) N-cadherin, (F) E-cadherin, (G) EpCAM, and (H) vimentin. Relative density was reported as mean ± SEM of 3–5 slides per treatment. (I–S) In combination with GEM, ASA and OP alter the stem cell population in pancreatic tumors. (M) Expression of CD36, (N) CD44, (O) CD24, (P) CXCR4, (Q) ratio CD44/24, (R) ALDH1 and (S) ABCG2 on pancreatic tumor tissue following treatment with ASA/OP/GEM. Tissue images were captured using the Nikon Eclipse EI R STG HNDL TRINOC microscope equipped with digital sight 1000 microscope camera using the 40× objective. Scale bars represent 200 μm. The one-way ANOVA Fisher test was used for comparison tests at 95% confidence intervals. The indicated asterisks represent the statistical significance compared to the untreated CTRL or otherwise shown. Abbreviations: ASA—aspirin; OP—oseltamivir phosphate; GEM—gemcitabine; CTRL—control; SEM—standard error of the mean.

Fig 4: Continuous perfusion of ASA and OP in combination with GEM inhibits pancreatic tumor xenograft cell differentiation and proliferation and promotes apoptosis in RAG2xCγ double mutant mice. (A) Expression of pancreatic xenograft tumor cell differentiation marker PDX-1 following treatment with ASA/OP/GEM. Tissue images were captured using the Nikon Eclipse EI R STG HNDL TRINOC microscope equipped with digital sight 1000 microscope camera using the 20× objective. Scale bars represent 100 μm. (B) Expression of proliferation marker Ki67 following treatment with ASA/OP/GEM and visualized using an epi-fluorescent microscope using the 40× objective. Scale bars represent 200 μm. (C) TUNEL assay was performed on sections of paraffin-embedded tumor tissue and visualized using an epi-fluorescent microscope using the 20× objective. Scale bars represent 100 μm. Quantification of the relative density of (D) PDX-1, (E) Ki67, (F) TUNEL, as a measure of the number of positive cells, (G) BCL2 and (H) c-myc. Relative density was reported as mean ± SEM of 3–5 slides per treatment. The one-way ANOVA Fisher test comparisons with 95% confidence, indicating asterisks for statistical significance compared to the untreated CTRL, unless otherwise shown. Data are presented as mean ± SEM of individual staining. Abbreviations: ASA—aspirin; OP—oseltamivir phosphate; GEM—gemcitabine; CTRL—control; SEM—standard error of the mean.

Fig 5: The study outline to evaluate the efficacy of constant perfusion of aspirin (ASA) and oseltamivir phosphate (OP) treatment at the end of pump perfusion (98 days post-pump implant) and the study endpoint (121 days post-pump implant). (A) Animals were divided into three cohorts: untreated control (CTRL), gemcitabine (GEM)-only treated, or implanted with an osmotic pump containing ASA (50 mg/kg) and OP (40 mg/kg) and treated weekly with GEM at the indicated dosages (B). Half of the animals in each cohort were euthanized at the end of the pump perfusion (day 98; mouse numbered in red) and the other half at the study endpoint (day 121, mouse numbered in black). Tumors, lungs, and livers were resected at necropsy and analyzed. (B) Measurement of tumor volume for each animal during the study. Pumps were implanted once each animal had a palpable tumor on day 52. The green square depicts the pump range. (C) Tumor wet weight at day 98 for each mouse and (D) Tumor wet weight at day 121 for each mouse. (E, F) Images of resected tumors at necropsy at the pump endpoints at days 98 and 121. (G) Body weight for untreated, GEM and PUMP (OP + ASA) days post tumor injection. (H) Quantification of metastatic nodules in the liver counted at the pump endpoint necropsy. (I) Gross histology of livers and metastatic nodules at necropsy at day 98. (J) Quantification of metastatic nodules counted at day 121 for the study endpoint necropsy. (K) Gross histology of livers and metastatic nodules at necropsy at day 121. (L) Quantifying the number of metastatic clusters counted from 2 + cuts of liver tissue at the end of the pump perfusion (day 98). (M) H&E staining of liver tissue at the pump endpoint. (N) Quantifying the number of metastatic clusters counted from 2 + cuts of liver tissue at the study endpoint (day 121). (O) H&E staining of liver tissue at the study endpoint. (P) Weight of livers at necropsy at day 98 and (Q) day 121. (R) Gross histology of lungs at necropsy at day 98, (S) Quantification of metastatic clusters in the lung from 2 + cuts of lung tissue at day 98, the pump endpoint necropsy, and (T) Lung weight at day 98. (W) Gross histology of lungs at necropsy at day 121. (X) Quantifying the number of metastatic clusters counted from 2 + cuts of lung tissue at the study endpoint day 121, (Y) Lung weight at day 121. H&E slides were visualized using the Nikon Eclipse EI R STG HNDL TRINOC microscope equipped with digital sight 1000 microscope camera with 20× objective. The scale bar represents 100 μm. Abbreviations: H&E—hematoxylin and eosin.