Anti-tumor immune responses following neoadjuvant immunotherapy with a recombinant adenovirus expressing HSP72 to rodent tumors

Abstract

Gene modification of tumor cells is commonly utilized in various strategies of immunotherapy preventive both as treatment and a means to modify tumor growth. Gene transfer prior to surgery as neoadjuvant therapy has not been studied systematically. We addressed, whether direct intra-tumoral injection of a recombinant adenovirus expressing the immunomodulatory molecule, heat shock protein 72 (ADHSP72), administered prior to surgery could result in sustainable anti-tumor immune responses capable of affecting tumor progression and survival in a number of different murine and rat tumor models. Using intra-dermal murine models of melanoma (B16), colorectal carcinoma (CT26), prostate cancer (TrampC2) and a rat model of glioblastoma (9L), tumors were treated with vehicle or GFP expressing adenovirus (ADGFP) or ADHSP72. Tumors were surgically excised after 72 h. Approximately 25-50% of animals in the ADHSP72 treatment group but not in control groups showed sustained resistance to subsequent tumor challenge. Tumor resistance was associated with development of anti-tumor cellular immune responses. Efficacy of ADHSP72 as neoadjuvant therapy was dependent on the size of the initial tumor with greater likelihood of immune response generation and tumor resistance associated with smaller tumor size at initial treatment. ADHSP72 neoadjuvant therapy resulted in prolonged survival of animals upon re-challenge with autologous tumor cells compared to ADGFP or vehicle control groups. To study the effects on tumor progression of distant metastases, a single tumor focus of animals with multifocal intra-dermal tumors was treated. ADHSP72 diminished progression of the secondary tumor focus and prolonged survival, but only when the secondary tumor focus was <50 mm3 . Our results indicate that gene modification of tumors prior to surgical intervention may be beneficial to prevent recurrence in specific circumstances.

Fig. 1

Transduction efficiency of murine and rodent tumor cell lines with recombinant adenovirus. a ADGFP was added at various multiplicities of infection (MOI; see legend) to cell lines grown to 70% confluency in 35-mm tissue culture dishes in DMEM-2% FCS for 90 min followed by two washes in phosphate buffered saline (PBS) and then grown overnight in complete media. After 24 h fluorescent cells were counted on a hemacytometer using a fluorescence microscope. The percent fluorescent cells as a fraction of total cell number is given on the y-axis. Data represent results from a typical experiment. Infections were performed in triplicate. b Intra-dermal tumors of either 100 mm³ or 200 mm³ were injected with 5 × 10⁸ pfu ADGFP in total volume of 100 ul PBS. Tumors were surgically excised after 72 h and digested in triple enzyme solution and placed in culture. Cells were evaluated for fluorescence as previously described. c Intra-dermal tumors were injected numerous times (see legend) with 5 × 10⁸ pfu ADGFP in total volume of 100 ul PBS at 24 h intervals. Tumors were surgically excised after 72 h and digested in triple enzyme solution and placed in culture. Cells were evaluated for fluorescence as previously described

Fig. 2

Western blot analysis of HSP72 expression. Immunoblot of CT26 cells infected at increasing multiplicity of infections with ADHSP72 were analyzed by anti-HSP72 antibody (SPA-810; Stress gen). Equivalent protein concentrations were loaded in each well

Fig. 3

CTL activity from ADHSP72 treated, tumor resistant animals. Percent specific lysis of target cells by 4-h ⁵¹Cr- release assay is depicted on the y-axis. The splenic effector:target cell cell ratio is given on the y-axis. Effector:target incubations were performed in triplicate. Animals with intra-dermal CT26 tumors were treated with ADHSP72 and re-challenged with CT26 cells 14 days after surgical excision of the primary tumor. Tumor resistance was determined by the lack of palpable tumor 14 days after secondary CT26 cell inoculation. a CTL activity from pooled splenocytes from two animals resistant to tumor challenge is depicted. b CTL activity from pooled splenocytes from two animals non-resistant to tumor challenge is depicted

Fig. 4

CTL activity from ADHSP72 treated, tumor resistant animals. Percent specific lysis of target cells by 4-h ⁵¹Cr- release assay is depicted on the y-axis. The splenic effector:target cell cell ratio is given on the y-axis. Effector:target incubations were performed in triplicate. Animals with intra-dermal B16 tumors were treated with ADHSP72 and re-challenged with B16 cells 14 days after surgical excision of the primary tumor. Tumor resistance was determined by the lack of palpable tumor 14 days after secondary B16 cell inoculation. a CTL activity from pooled splenocytes from two animals resistant to tumor challenge is depicted. b CTL activity from pooled splenocytes from two animals non-resistant to tumor challenge is depicted

Fig. 5

CTL activity from ADHSP72 treated, tumor resistant animals. Percent specific lysis of target cells by 4-h ⁵¹Cr- release assay is depicted on the y-axis. The splenic effector:target cell cell ratio is given on the y-axis. Effector:target incubations were performed in triplicate. Animals with intra-dermal TrampC2 tumors were treated with ADHSP72 and re-challenged with TrampC2 cells 14 days after surgical excision of the primary tumor. Tumor resistance was determined by the lack of palpable tumor 14 days after secondary TrampC2 cell inoculation. a CTL activity from pooled splenocytes from two animals resistant to tumor challenge is depicted. b CTL activity from pooled splenocytes from two animals non-resistant to tumor challenge is depicted

Fig. 6

CTL activity from ADHSP72 treated, tumor resistant animals. Percent specific lysis of target cells by 4-h ⁵¹Cr- release assay is depicted on the y-axis. The splenic effector:target cell cell ratio is given on the y-axis. Effector:target incubations were performed in triplicate. Animals with intra-dermal 9L tumors were treated with ADHSP72 and re-challenged with 9L cells 14 days after surgical excision of the primary tumor. Tumor resistance was determined by the lack of palpable tumor 14 days after secondary 9L cell inoculation. a CTL activity from pooled splenocytes from two animals resistant to tumor challenge is depicted. b CTL activity from pooled splenocytes from two animals non-resistant to tumor challenge is depicted

Fig. 7

CTL Assays with blocking antibodies. Percent specific lysis of target cells by 4-h ⁵¹Cr- release assay is depicted on the y-axis. Pooled splenocytes from two animals resistant to autologous tumor challenge from the treatment groups identified on the x-axis at an effector to target ratio 100:1. Anti-CD4 or anti-CD8 antibodies (Pharmingen) were added at 30 ul/well to inhibit activity

Fig. 8

Survival after neoadjuvant treatment. a Neoadjuvant treatment of animals with intra-dermal CT26 tumors was performed as previously described with the vectors described in the legend. Tumors were surgically excised and animals were inoculated with CT26 cells 14 days after surgical excision and followed for survival. Criteria for removal of animals from consideration included weight loss>12 %, inability to ambulate, inability to feed or loss of normal grooming. Data are representative of one of three independent experiments with similar results. (*, P<0.02) denotes statistical significance by Kaplan-Meier survival analysis. b Neoadjuvant treatment of animals with intra-dermal B16 tumors was performed as previously described with the vectors described in the legend. Tumors were surgically excised and animals were inoculated with B16 cells 14 days after surgical excision and followed for survival. Data are representative of one of three independent experiments with similar results. (*; P<0.02) denotes statistical significance by Kaplan-Meier survival analysis

Fig. 9

Effect of neoadjuvant immuno-gene therapy on distant tumor growth and survival. Animals (n=10/group) were inoculated with B16 cells on opposite flanks, 5 days apart. Animals were grouped based on the size of the secondary tumor foci at the time of injection of the primary tumor. a Tumor progression (solid lines) and survival (dotted lines) of animals with secondary tumor foci < 50 mm³ when the larger tumor was injected with PBS (open circle, filled circle), ADGFP (open triangle, filled triangle) or ADHSP72 (open square, filled square) and surgically excised after 72 h. b Tumor progression (solid lines) and survival (dotted lines) of animals with secondary tumor foci > 50 mm³ when the larger tumor was injected with PBS (open circle,filled circle), ADGFP (open diamond,filled triangle) or ADHSP72 (open square,filled square) and surgically excised after 72 h. (*) denotes statistical significance by ANOVA