Monday, November 28, 2011

Combination Therapy--Giving Cancer the 1-2 Punch

It has been well documented that many cancer cells over express Intreleukin-13 receptors—in particular 4T1 breast carcinoma cell lines and MCA304 sarcoma cell lines (Joshi, 2000). Interleukin-13 (IL-13) is a small, cell-signaling protein that is derived from a Th2 immune response that binds to Interleukin-13 receptors (IL-13R). IL-13Ralpha2 is one of two receptor subunits of the IL-13R complex. This subunit binds IL-13 with high affinity to mediate a signal transduction, ultimately resulting in the production of TGF-beta (Fichtner-Feigl et al., 2006).
Recent studies have focused on targeting IL-13R of tumor cells in order to control the progression of cancer. For this reason, an immunotoxin, IL13-PE, was formulated by recombining IL-13 and Pseudomonas exotoxin in order to target IL-13R expressing tumor cells (Kawakami et al., 2001). IL13-PE is able to specifically recognize IL-13Rs, limiting toxicity to cancerous tissues (Puri et al., 1996). IL13-PE acts to inhibit protein synthesis and cause cell death of IL-13 expressing cells by irreversibly ADP-ribosylating the diphthamide residue of elongation factor 2 (Fichtner-Feigl et al., 2006).
Vaccines have also been utilized as an alternative method to induce a strong immune response against tumor cells. T cells and B cells are leukocytes that are responsible for regulating the immune response by recognizing harmful entities in the body and carrying out their destruction. Antitumor vaccines are intended to incur an antigen-specific immune response against a tumor peptide by priming the immune system to activate T cells and B cells (Nakashima et al., 2010). By stimulating the production of T cells and B cells to recognize tumor cells, the proliferation of tumors can be controlled. One particular vaccine, IL-13Ralpha2 DNA vaccine, has shown to be effective in limiting tumor growth in both 4T1 breast carcinoma cell lines and MCA304 sarcoma cell lines (Nakashima et al., 2010).

With confirmation that either IL13-PE or the IL-13Ralpha2 DNA vaccine individually limits tumor cell growth, Nakashima et al. progressed to test the effects of tumor progression in mice treated with both, termed combination therapy. They found combination therapy to be very successful, with 33% of mice in MCS304 and 4T1 tumor models having complete responses. The mechanisms behind the tumor regression were analyzed, and the response can be attributed to, in part, by infiltration of CD8+ and CD4+ T cells and the elimination of MDSCs (Nakashima et al., 2011).
IL13-PE was injected into the tumor 7, 9, and 11 days after tumor development and IL13Ralpha2 DNA vaccine was injected on days 13, 18, 23, and 28. Mice with this combination treatment showed continual tumor reduction during the treatment in both 4T1 and MCA304 cell lines (Nakashima et al., 2011). From these results, it was shown that combination therapy was very successful, and its results were then further compared to the individual effects of IL13-PE alone and IL-13Ralpha2 DNA vaccine when used alone.
IL13-PE alone was then administered to mice with established tumors in the same manner as in the combination therapy, except the mice were injected with a mock vaccine in the place of IL-13Ralpha2 vaccine. It was found that treatment with IL13-PE would delay tumor growth compared to an untreated tumor, but after 21 days of tumor development, the tumor began to grow again. Similar results were obtained in both 4T1 and MCA304 cell lines (Nakashima et al., 2011).
The size of tumors after 30 days in mice undergoing combination therapy was much smaller than in either method of tumor reduction alone. This was demonstrated by the time it took until tumors reached 2 cm in diameter. Untreated mice injected with PBS and VR mock-vaccine, reached the 2 cm mark after 30 and 31 days. IL13Ralpha2 DNA vaccine alone group reached 2 cm in diameter after 40 days. IL13-PE alone group reached 2 cm in diameter after 45 days. Mice undergoing combination therapy took the longest for tumors to reach 2 cm in diameter, with 65 days. Combination therapy resulted in over a two-fold increase in time before tumor maximum size was reached compared to untreated mice. Also, two of the six mice in both the MCA304 and 4T1 models showed a complete response with tumor-free survival (Nakashima et al., 2011). This experiment proved that both the IL13-PE alone or IL13Ralpha2 DNA vaccine alone would result in delayed tumor growth, but combination therapy was more effective in reducing tumor development time and increasing chance of survival in 4T1 and MCA304 tumor-bearing mice.
4T1 breast tumor is reportedly highly metastatic (Ostrand-Rosenberg et al., 1996). Lymph nodes were harvested on 30 days after tumor development to test the effect of combination therapy on metastasis. Mice receiving combination therapy had significantly lower numbers of tumor nodules in the untreated, PBS control, IL13-PE alone, and IL-13Ralpha2 DNA vaccine alone (Nakashima et al., 2011). From this experiment, it was concluded that combination therapy also had an effect on the metastatic potential of a tumor.
Nakashima et al. hypothesized that cytotoxic T lymphocytes, or CTLs, are responsible for the observed effects of combination therapy against 4T1 and MCA304 tumors. CTLs are a subset of white blood cells that patrol the body and are responsible for recognizing foreign entities, sequestering them, and carrying out their destruction. Nakashima et al. harvested splenocytes on day 30 after tumor appearance and restimulated them with mitomycin-c-treated 4T1 tumor cells or CT-26 as a negative control for 1 week. The negative control showed that there was no lysis from CTL activity. It was found that there was significantly greater CTL activity for the IL13-PE alone group (17 +/-1%) than that of IL-13Ralpha2 DNA vaccine alone group (10 +/- 2%).
With confirmation that CTL activity was increased, Nakashima et al. examined the possible underlying mechanisms by determining if there was an increase in IFN-gamma, CD4+ T cells, or CD8+ T cells. IFN-gamma is a molecule produced by many immune cells that promotes the proliferation of immune cells in response to a foreign entity, or directs immune cells to areas in need. The release of IFN-gamma was determined by analyzing splenocytes. The untreated, PBS control showed that untreated mice had low levels of IFN-gamma, releasing only 206 pg/ml. Similarly, the negative controls CT-26 and D5 tumor-bearing mice released no or low level of IFN-gamma. However, IFN-gamma release was significantly increased in IL13-PE or IL13Ralpha2 DNA vaccine treated cells. Treatment with IL13-PE alone resulted in a release of 792 +/- 136 pg/ml of IFN-gamma, and the IL13Ralpha2 DNA vaccine alone released 971 +/- 153 pg/ml of IFN-gamma. Combination therapy produced an even larger response with 1249 +/- 93 pg/ml of IFN-gamma released. Similar results observed in MCA304 tumor model. From this observed increase in IFN-gamma in mice treated with both IL13-PE and IL13Ralpha2, it can be concluded that a specific CTL response was either induced or amplified and IFN-gamma release may be responsible for delaying tumor growth.
CD4+ and CD8+ T cells levels were then measured to determine if they were responsible for the delayed tumor growth associated with combination therapy. CD4+ and CD8+ T cells are immune cells that are capable of recognizing foreign entities and killing them. Samples were collected 30 days after tumor development, marked using specific antibodies, and viewed with immunofluorescence microscopy to determine presence or absence of the T cells. Few CD4+ T cells were present in the untreated control, with 13 +/- 2 CD4+ T cells observed. There were increased numbers in mice treated with either IL13Ralpha2 DNA vaccine or IL13-PE, with 29 +/- 4 and 31 +/- 6 CD4+ T cells observed, respectively. An even more drastic increase was observed in samples that were treated with combination therapy, with 53+/- 4 CD4+ T cells.
Similar findings were also reported for CD8+ T cells. In the untreated samples, 19 +/- 2 CD8+ T cells were observed. Again, there was an increase in the treated samples, with IL13Ralpha2 DNA vaccine alone having 59 +/- 3 CD8+ T cells and IL13-PE alone having 51 +/-3 CD8+ T cells. Finally, when using combination therapy, there was an even more impressive quantity, with 119+/-8 CD8+ T cells observed. From these results, it was found that the number of CD4+ and CD8+ T cells increased with combination therapy almost twice as much as when treated with either IL13Ralpha2 DNA vaccine or IL13-PE individually.
With confirmation of increased numbers of T cells and increased IFN-gamma, Nakashima et al. continued to assess the changes in concentration of chemokines. Chemokines are responsible for controlling the migration of immune cells to the site of the foreign entity in the body. CXCL9 and CXCL10 are chemokines that have been proven to be involved in tumor regression (Di Carlo et al., 2004). Furthermore, the majority of activated T lymphocytes express CXCR3 (Qin, 2004). From staining with antibodies in 4T1 and MCA304 tumor cell lines, it was found that mice receiving combination therapy tested positive for CXCL9 and CXCL10, while the control, untreated mice tested negative. CXCR3, CXCL9, and CXCL10 were all highly expressed in combination therapy treated mice compared to either IL13-PE or IL13Ralpha2 DNA vaccine alone treated mice.
CD4+ and CD8+ T cells were then depleted from samples to assess if they were the critical factors in delaying tumor growth. Using specific antibodies, over 95% of all CD4+ T cells, CD8+ T cells, and Natural Killer cells were depleted, confirmed by cytometric analysis. It was found that depletion of CD4+ and CD8+ T cells completely negated all effects normally observed combination therapy, resulting in tumor growth resembling untreated tumors (1335 +/- 272 mm3 vs 1474+/-104 mm3) on day 21. CD8+were determined to be critical for the antitumor response, for depletion of CD8+ T cells almost completely negated the effect of combination therapy. It was determined that natural killer cells had little to no purpose in the immune response against a tumor, for depleting natural killer cells had no effect on tumor growth or regression. From this experiment, it was determined that CD8+ T cells are the critical immune cells that are stimulated by combination therapy and are responsible for the delayed growth of tumors, with help from CD4+ T cells, but not natural killer cells.
It was further confirmed that combination therapy acts to limit tumor progression by the means of T cells by using RAG-2 deficient mice. RAG-2 is required in the production of B and T cells. Without RAG-2, the immune system would be devoid of B and T cells. Combination therapy administered to RAG-2 deficient mice resulted in tumor growth comparable to untreated mice, with treated tumor sizes of 1861+/-197 mm3 and untreated tumor sizes of 1820 +/- 287 mm3 27 days after tumor development. Therefore, T and B cells must be involved in the antitumor response combination therapy evokes.
Antibody levels against IL-13Ralpha2 were measured in blood serum samples collected 33 days after tumor development in both 4T1 and MCA304 tumor cell lines. In the 4T1 tumor model, the amount of antibody was found to be low in the untreated mice, with 118 +/- 51 ng/ml. Antibody amounts were increased in both IL-13Ralpha2 DNA vaccine alone mice, with 290 +/- 82 ng/ml of antibody present, and in IL 13-PE alone with 328+/-78 ng/ml. Moreover, antibody amounts were significantly increased in mice receiving combination therapy, with 635 +/- 40 ng/ml of antibody present in the blood serum. Similar results were observed in MCA304 tumor cell lines.
It was hypothesized that combination therapy was also limited tumor growth from altering number of regulatory T cells (Tregs). Tregs are cells that are responsible for controlling immune cell proliferation and dampening an immune response. Certain types of cancers have been related to Tregs, which dampen the immune response against a tumor (Nomura and Sakaguchi, 2005). Splenocytes were harvested 30 days after tumor development and stained for Treg markers CD3, CD4, CD25, and Foxp3. Nakashima et al. found that 7.5% of cells in splenocytes of tumor-free mice were Tregs, a relatively low number. There was a significant increase in the Treg population in tumor-bearing mice with no treatment, at 17%. Tregs in IL-13Ralpha2 DNA vaccine alone treatment and IL13-PE alone treatment were at intermediate levels, 10% and 12%, respectively. Combination therapy, on the other hand, resulted in a significant reduction of Tregs to levels comparable to tumor-free mice, with 8%.
Nakashima et al. further assed number of Tregs infiltrating into tumors by immunofluorescence microscopic analysis. Tumors were harvested from each group 30 days after tumor development and stained for Treg markers on the cell surface. All three treatments decreased Treg numbers compared to the untreated tumor, which had 51% of cells being Tregs. IL13-PE alone treated samples had 32% and IL-13Ralpha2 DNA vaccine treated samples had 33%, showing decrease in each individual treatment. Most notably, combination therapy had 11.5% of cells as Tregs. These results demonstrate that treatment with either IL13-PE or IL-13Ralpha2 DNA vaccine would decrease the amount of Tregs within the tumor and within the spleen, but combination therapy had even more drastic Treg reductions.
Nakashima et al. further assessed the level of myeloid-derived suppressor cells, or MDSCs. MDSCs are important in tumor development for they are immune cells that inhibit T cell responses, which play a key role in the inability to recognize and destroy a tumor (Allan, 2008). MDSC levels were measured in splenocytes. Mice without tumors had low levels of MDSCs. 30 days after tumor development, it was found that mice with tumors and no treatment had 45% of MDSCs. 12 days after tumor development, IL13-PE treated mice showed a reduction with 25% of MDSCs. Similarly, IL-13Ralpha2 DNA vaccine treated mice showed a reduction with 38% of MDSCs. Combination therapy resulted in an even greater reduction, with only 10% of MDSCs. Therefore, combination therapy resulted in a lower number of infiltrating MDSCs compared with untreated tumors as well as with treatment with either IL13-PE alone or IL-13Ralpha2 DNA vaccine alone. From this data, it can be inferred that combination therapy decreased immunosuppressive MDSCs in spleen and tumor, which could further enhance the efficacy of combination therapy (Nakashima et al., 2011).
From the experiments of Nakashima et al., much has been learned about a potential method to limit tumor growth and spread. IL13-PE and IL-13Ralpha2 DNA vaccine each delayed the progression of tumor growth when applied individually, but when combined had a synergistic effect with overwhelmingly significant results. It was most notable that in using combination therapy, 33% of the mice treated had complete responses and survived. Nakashima et al. also helped to shed light on the mechanisms underlying the delay of tumor growth. CD8+ T cells, along with CD4+ T cells, T regulatory cells and IFN-gamma each played a part in the regression of tumors. Altered numbers of these cells and products were responsible for tumor regression through CTL activation. Also, it was shown that combination therapy successfully inhibited the lunch and lymph node metastasis as well as eliminating MDSCs. It would be interesting if this research was extended to explore the effects of combination therapy on other tumor cell lines other than 4T1 and MCA304, for IL13R’s are commonly expressed receptors that are associated with a wide variety of cancers. This research may provide key insight on how to maintain and control tumor growth and potentially treat tumor developments by boosting the immune system by targeting IL13R’s.
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