Synergistic Effects Of Conventional Chemotherapy And Immunotherapy Of Cancer Is Mediated Through M6PR

Objective1. To confirm the potentiation of the antitumor effects of immunotherapy in combination with chemotherapy by the investigation of different kinds of tumors established in mice. To test the effect of chemotherapy on M6PR in tumor cells by the treatment of B16F10,4T1or U266tumor cells in vitro and their tumors established in mice in vivo with chemotherapy. To test the role of M6PR in the combined therapy in vivo wild-type (H2Kb+/+) B16F10cells expressing luciferase (B16-Luc) and B16F10H2Kb-/-cells (B16-H2Kb-) were used. B16-H2Kb-cells were transfected with control or MPR shRNA and their tumors established in mice were treated with different therapy.2. To investigate the mechanism of MPR up-regulation by chemotherapy through detecting the mpr mRNA and its protein expression in B16F10,4T1or U266cells in vitro and their tumors established in mice in vivo with chemotherapy. To study the association between MPR up-regulation and the expression of IGF-Ⅱ in tumor cells treated with chemotherapy. To test the in tumor cells induced by chemotherapy in vitro. To test the hypothesis of potentiating antitumor effects of immunotherapy combined with chemotherapy through investigating the re-distribution of M6PR in tumor cells by the up-regulation or down-regulation of autophagy.Methods1. The expression of M6PR on the surface of B16F10,4T1or U266tumor cells after chemotherapy was evaluated by flow cytometry. The M6PR protein levels in B16F10,4T1or U266tumors established in mice after chemotherapy were evaluated by Immunohistochemistry. wild-type (H2Kb+/+) B16F10cells expressing luciferase (B16-Luc) and B16F10H2Kb-/-cells (B16-H2Kb-) were used. B16-H2Kb-cells were transfected with control or MPR shRNA and their tumors established in mice were treated with different therapy. Tumor growth was monitored three times a week by caliper measurement and twice a week by in vivo imaging.2. The mpr mRNA level in B16F10,4T1or U266cells in vitro and in vivo after chemotherapy was evaluated by Real-time PCR. The total M6PR protein and membrane fractions in these tumor cells were detected by Western blotting. The IGF-II protein level in tumor cells treated with chemotherapy was evaluated by Western blotting in order to study the mechanism of MPR up-regulation by chemotherapy. The autophagy in tumor cells induced by chemotherapy was evaluated by confocal microscopy. The re-distribution of M6PR in tumor cells after chemotherapy was detected using confocal microscopy and flow cytometry through the blockade of autophagy with either its inhibitor3MA or down-regulating atg5and through the up-regulation of autophagy with Rapamycin.Results1. The antitumor effect of immunotherapy combined with chemotherapy In MC38colon carcinoma models, Vaccination of mice with DCs transduced with adenovirus containing full-length mouse wild-type p53(Ad-p53) resulted in a substantial delay in tumor progression compared with no treatment (P<0.05). Treatment of mice with TAX alone delayed tumor growth, but tumor progression resumed soon after the treatment was discontinued. The combination of TAX and the DC vaccine potently suppressed tumor growth compared with either immunotherapy or chemotherapy alone (P<0.05). In TUBO breast carcinoma models, single TAX had very little antitumor activity and vaccination alone only slightly delayed tumor growth compared with no treatment (P>0.05). The combination of the DC vaccine with TAX treatment resulted in a substantial antitumor effect in this model as well (P<0.05).In EG-7lymphoma models, T cells or TAX alone caused a decrease in tumor growth compared with no treatment (P<0.05). This effect was substantially more pronounced when T cell transfer and TAX administration were combined (P<0.05).2. The effect of chemotherapy on M6PR on tumor cell surface(1) Several mouse (B16F10melanoma,4T1mammary carcinoma) and human multiple myeloma (8226, H929, U266) tumor cell lines were used to test the effects of chemotherapy on the expression of MPR. Different chemotherapeutic agents caused substantial and similar up-regulation of MPR expression in vitro in all tested mouse and human tumor cell lines (P<0.05). Chemotherapy also increased granzyme B (GrzB) penetration into cells (P<0.05).In B16F10,4T1tumor models significant (P<0.05) up-regulation of MPR expression was detected48hr after the injection of TAX using immunohistochemistry. It remained elevated for another24hr and then returned to the pre-treatment level within5days after injection. It also happened in U266tumors in nude mice treated with DOX.(2) In B16F10tumor bearing mice, administration of either Pmel-1CTLs or TAX alone substantially delayed tumor growth, which however, resumed one week after the treatment. When TAX was combined with T-cell therapy, a significant (P<0.05) potentiating effect was observed. However, this effect was seen only if TAX was administered two days after T cells. When TAX was injected5days prior to T-cell administration, no increased antitumor effect was observed.3. The role of M6PR in the effect of immunotherapy combined with chemotherapy(1) In B16F10control shRNA tumor bearing mice, Pmel-1CTLs combined with TAX resulted in an increased antitumor effect compared with single therapy (P<0.05). In B16F10M6PR shRNA tumor bearing mice, no increased antitumor effect was observed in combination therapy (P>0.05).(2) In B16F10M6PR shRNA Kb-tumor bearing mice, single Trp2180-188CTLs did not show any antitumor effect compared with no treatment (P>0.05). No increased antitumor effect was observed in combination therapy compared with single TAX (.P>0.05). In B16F10control shRNA Kb" tumor bearing mice, no increased antitumor effect was observed in combination therapy (P>0.05).(3) B16-Luc and B16-control shRNA Kb-tumors were established in the opposite flanks (B16-Luc-left flank; B16-control shRNA Kb--right flank) of the same mouse. Significant (P<0.05) potentiation of the antitumor effects of combined TAX and CTLs was observed only in the flank with B16-Luc tumor but not with B16-control shRNA Kb" tumors. In the B16-Luc mixed with control shRNA Kb-tumor bearing mice, combining TAX and CTLs resulted in a significantly (P<0.05) greater antitumor effect than each of them separately. This was seen by monitoring tumor size with calipers and by in vivo imaging. In the B16-Luc mixed with M6PR shRNA Kb-tumor bearing mice, potentiation of the antitumor effect of combination therapy was not detected (P>0.05) while TAX and CTLs combined had potent antitumor activity against B16-Luc cells when these cells were monitored by in vivo imaging (P<0.05).4. The effect of chemotherapy on MPR synthesis, degradation and re-distribution(1) The result of Real-time PCR showed that no effect of chemotherapy drugs on mpr mRNA expression was found in B16F10,4T1and EL-4tumor cells (P>0.05). Treatment of B16F10or4T1tumor cells in vitro with TAX resulted in up-regulation of MPR on the cell surface (P>0.05). However, when the total amount of MPR was evaluated using Western blot, no effect of TAX or DOX on MPR was found. Similar results were obtained when cells were analyzed by flow cytometry after fixation and permeabilization prior to staining with anti-MPR antibody (P>0.05).In these tumor cell lines treated with TAX or DOX, chemotherapy induced substantial accumulation of MPR only in a membrane fraction (P<0.05).(2) We evaluated MPR expression in human MM U266cells treated with DOX using confocal microscopy. Chemotherapy caused redistribution of MPR from a primarily cytoplasmic to a predominantly membrane localization of MPR. We assessed the proportion of MM cells with predominantly membrane localization of MPR using bone marrow slides from MM patietns. Prior to treatment only20%of the MM cells had such a characteristic, whereas after3days of high-dose chemotherapy this proportion increased to more than50%of all MM cells (P<0.05).5. The effect of autophagy on the re-distribution of M6PR in tumor cells(1) The flow data showed that incubation of B16F10tumor cells with recombinant IGF-Ⅱ led to the loss of the tumor cell’s ability to up-regulate MPR expression on the surface in response to TAX. This was associated with loss of TAX-inducible up-regulation of GrzB uptake by tumor cells (P<0.05). The effect of chemotherapy on4T1, EL4. and B16F10tumor cell lines in vitro were evaluated by Western blotting. In all cases no decrease in IGF-II expression was detected (P>0.05). We also tested the in vivo effect of TAX treatment in4T1and B16F10tumor-bearing mice. Chemotherapy treatment caused a substantial increase in the level of IGF-2(P>0.05).(2) Treatment of tumor cells with TAX, CIS, or DOX caused rapid induction of autophagy as determined by the appearance of autophagy-specific LC3punctae in treated cells by confocal microscopy. Inhibition of autophagy with3MA abrogated up-regulation of MPR on the surface of U266MM cells treated with DOX or B16F10cells treated with TAX (P<0.05). Transfection of B16F10cells with atg5siRNA completely abrogated the TAX-inducible increase in MPR expression (P<0.05). Rapamycin caused a substantial increase in MPR levels on the tumor cell surface (P<0.05). B16F10tumor cells were transfected with LC3-GFP. After treatment of the cells with TAX, LC3punctae became easily visualized by confocal microscopy. When untreated cells were stained with MPR antibody, no co-localization of MPR and LC3were seen. In contrast, after TAX, co-localization of autophagosomes and MPR was readily detectable.ConclusionCancer immunotherapy in combination with chemotherapy may produce an increased antitumor effect. The antitumor effect of CTL is associated with endocytosis induced by M6PR. Chemotherapy combined with immunotherapy results in bystander killing of tumor cells without the need for antigen recognition and this effect was mediated by MPR. Chemotherapy dose not induce MPR synthesis or inhibit its degradation. Changes in IGF-2levels caused by chemotherapy were not associated with up-regulation of MPR on the tumor cell surface. Chemotherapy-inducible autophagy causes up-regulation of MPR on tumor cell surface.