ainst AE17sOVA, mice received 3 vaccine doses (107 cells/dose s.c.) one week apart, prior to tumour challenge. Multiple vaccinations with heat-shocked cell lysate had no added benefit on survival over a single vaccination, and multiple vaccinetions with untreated cell lysate were still unable to promote survival (Figure 2(a)). Emergence of tumour in unprotected mice receiving multiple doses of heat-shocked cell lysate was also significantly delayed (~20 days; Figure 2(b)). However, unlike tumour kinetics in the singledose experiments, once tumours did emerge they appeared to grow more rapidly than in saline or untreated cell lysate vaccinated mice (Figure 2(b)).

3.4. Both Heat-Shocked and Non Heat-Shocked Lysate Vaccines Induce Maturation of BMDC

To determine if the increased potency of HSL vaccination was due to adjuvant effects on DCs we measured the level of maturation induced by HSL and lysate on BMDC in-vitro, BMDCs were stimulated 5 to 1 with equivalent tumour cells for 24 hr and then stained for MHC and co-stimulatory molecules. Unstimulated BMDC had relatively low levels of CD80, CD86 and MHC

Table 1. Effect of dose in the efficacy of lysate and heat shocked lysate vaccines.

(a)(b)

Figure 2. Multiple HSL vaccination does not improve resistance to tumour challenge. (a) Naïve C57Bl/6 mice were vaccinated subcutaneously with three doses of either untreated or heat-shocked cell lysates on days 0, 7 and 14 (1 × 107 cells/dose) prior to subcutaneous challenge with viable AE17sOVA tumour cells on day 28. No additional survival benefit was observed for mice receiving multiple vaccinations versus those that received single vaccination; (b) Mice that ultimately failed to respond heat shocked lysate vaccination demonstrated significantly delayed tumour growth relative to saline and untreated lysate controls; although once tumour began to develop it did so at a much faster rate relative to control mice.

class II (Figure 3(a)). Stimulation with lysate caused a marked upregulation in expression of all three surface markers. Stimulation with HSL also induced upregulation of CD80, CD86 and MHC class II but responses were not higher than with normal lysate.

3.5. Heat-Shocked AE17sOVA Lysate Induces Local DCs to Preferentially Migrate to Draining Lymph Node

We next investigated the kinetics involved in DC trafficking to lymph nodes, subsequent to exposure to HSL. Mice were vaccinated s.c. with 1 × 107 HSL followed 8 h later by treatment of the injection site with cell tracker blue dye in order identify migrating DCs. HSL-vaccinated mice showed a four-fold increase in the proportion of cells trafficking from the vaccine-site in the draining lymph node compared to those treated with lysate, and over a seven-fold increase compared to saline treated mice (Figure 3(b)). Trafficking cells were CD11c+ and CD8 (Figure 3(b)) and expressed CD11b and Dec205 (data not shown) consistent with a migratory DC profile [20]. Together, these data indicate that HSL and lysate vaccines can induce DC activation but that HSL treatment promotes superior recruitment of activated DCs in the draining lymphnodes.

3.6. Prophylactic Vaccination Promotes Long Term Immunological Protection

We next tested whether HSL vaccination could promote long term immunological protection (i.e. memory) against AE17sOVA tumour. Surviving mice from single vaccination experiments (Table 1, n = 4) were subcutaneously rechallenged on the contra-lateral flank with 1 × 106 viable tumour cells, 100 days after the initial tumour challenge (i.e. d14 + 100). On the same day, naïve control mice were inoculated with 1 × 106 AE17sOVA cells, having received saline vaccination 14 days earlier. Control mice succumbed to tumour growth by day 30, post tumour challenge, while 100% of HSL vaccinated mice survived tumour rechallenge with no visible tumours (Figure 4).

3.7. Therapeutic Intratumoural Injection of HSL Vaccine Delays Tumour Growth, but Does Not Protect against Tumour Challenge

To determine whether AE17sOVA HSL vaccination could protect against tumour challenge in a therapeutic setting, groups of 5 mice were first inoculated s.c. on day 0 with 1 × 106 viable AE17sOVA cells and then treated on day 10 with either AE17sOVA HSL or untreated AE17s OVA lysate equivalent to 1 × 107 cells, or with saline (control). Tumour growth was consistent between all groups with all mice succumbing to tumours by day 35 when vaccines were administered subcutaneously (Figure 5(a)). Interestingly, when vaccines were administered intratumorally (i.t.), we observed a significant delay in tumour growth (p < 0.05; ≥1.3-fold) in HSL vaccinated mice relative to other groups. However, therapeutic i.t. vaccination failed to eliminate AE17sOVA tumour development (Figure 5(b)).

4. Discussion

Aggressive surgery with adjunct chemotherapy or radiotherapy is one treatment regimen used for malignant

(a)(b)

Figure 3. Heat shocked lysate promotes equivalent level of maturation as lysate-stimulated BMDC. (a) Bone marrow derived dendritic cells were stimulated in vitro with heat-shocked or non-heat-shocked AE17sOVA lysate (equivalent to 5:1 tumor cells/DC) of left unstimulated for 24 hrs prior to staining for surface marker expression of CD11c and the DC maturation markers CD80, CD86 and MHC class II. Dark grey shaded histogram = isotype control; light grey shaded histogram = unstimulated; grey line = untreated lysate; black line = heat shock treated lysate; (b) Vaccine-induced DC trafficking to lymphnodes. Mice were injected with either heat-shocked lysate or untreated lysate (both 107 cells/injection) for 8 h. Cell Tracker Blue dye was then added to the injection site and after a further 12 h the number of “dye” containing CD11c+ DCs in the draining lymph node determined by flow cytometry after first pooling lymphnodes from 5 mice per group.

Figure 4. Prophylactic vaccination promotes long term immunological protection against tumour rechallenge. Surviving mice from single vaccination experiments (Table 1, n = 4) were rechallenged with 1 × 106 viable AE17sOVA cells (s.c) on the contra-lateral flank 100 days after initial tumour challenge. Control mice (n = 2) received tumour challenge 14 days post saline vaccination. 100% of HSL vaccinated mice survived tumour rechallenge while all control mice succumb to tumour by day 30.

mesothelioma with the intent of prolonging life, especially in younger patients. Yet they are seldom curative. Most MM patients experience a high recurrence rate of local disease and succumb within 9 to 12 months of diagnosis [21] highlighting the need for improved adjuvant therapies that will target residual or metastatic disease and improve survival after surgery. Immunotherapies, such as tumour vaccines have emerged as a therapeutic option for the management of cancer patients. These vaccines have shown most potential when targeted toward relatively immunogenic cancers with known tumour antigens such as melanoma and prostate cancer [22, 23]. However, despite these limited clinical successes, cancer vaccines to date have not lived up to their potential as many cancers, such as MM, are poorly immunogenic and their tumour associated antigens (TAAs) remain uncharacterised. In the absence of known tumour antigens, the use of autologous tumour lysates have provided an alternative vaccine approach, as by definition they should contain all relevant TAAs within them (reviewed in [24]). Hyperthermic treatment of tumour lysates can enhance their immunogenic potential due to the

(a)(b)

Figure 5. Therapeutic vaccination fails to protect against tumour rechallenge. Naïve mice were inoculated subcutaneously with 1 × 106 viable AE17sOVA cells 10 days prior to receiving saline control, untreated lysate or heat shock lysate via subcutaneous (s.c) or intratumoral (i.t) vaccination. (a) Consistent tumour growth was observed between all groups with all subcutaneously vaccinated mice succumbing to tumour by day 35; (b) Intratumoral therapeutic vaccination with heat shocked lysate significantly delayed tumour development relative to control or subcutaneous vaccination groups, but failed to protect against overall tumour development.

ability of heat shock proteins to promote cross presentation of antigenic peptides [3-5], a necessary step for inducing anti-tumour immunity. In contrast to previous studies that utilised HSP-peptide complexes purified from tumour lysates [1,2,25], we investigated whether a simple heat-shock protocol could be used to enrich MM tumour cells with HSPs prior to lysis and to test whether HS treatment enhanced the immunogenicity of the resulting tumour lysate vaccine.

Our data demonstrate that HS treatment of tumour cells results in increased Hsp70 expression equivalent to that observed in human mesothelioma cells. When lysates made from these cells were used as vaccines, we observed significantly improved protection and delayed tumour development. These results are consistent with previous reports demonstrating the anti-tumour properties of tumour derived HSP-peptide complexes [1,2,5]. Interestingly, both HSL and untreated lysate were able to induce similar levels of BMDC maturation following vaccination, although HSL showed enhanced recruitment of local DCs to the draining lymph node (dLN) compared to untreated lysate. Cross presentation of tumour antigen by DCs to T cells is essential for the development of antitumor immunity and the increased DCs trafficking to the dLN plus the ability of HSPs to more efficiently chaperon antigenic peptides for presentation [5,25] may explain the enhanced immunogenicity of the HSL vaccine. This has been exploited in a recent study using DC vaccines fused with purified HSP complexes to enhance DC vaccination efficacy [26]. However, these protocols are generally labour intensive, and require substantial time commitments and cost associated with preparation of vaccine components. Our data demonstrates that a simple, inexpensive method of enriching HSP in tumour lysates is comparable to more costly and time consuming autologous lysate preparation methods. Importantly, we have previously demonstrated the feasibility of this preparation method for treating MM [27].

In the prophylactic setting, a single s.c dose of the HSL vaccine was sufficient to induce long term immunological memory and protect against subsequent tumour rechallenge. However, as with many tumour immuno-therapies the s.c HSL vaccine failed to protect if administered therapeutically. Importantly, when HSL was used as a therapeutic vaccine administered intratumourally it was able to significantly delay tumour growth. Thus directly delivered HSL vaccines could boost local anti-tumor responses, at possibly the most relevant site. We observed a similar result in an earlier study using our MM model following i.t delivery of interleukin-2 (IL-2) plus anti-CD40 combined adjuvant immunotherapy [28]. Here, i.t. delivered IL-2 + anti-CD40, induced local inflammation that prompted tumour destruction and release of autologous TAA, which in turn promoted the development of a systemic anti-tumour immune response. These results demonstrate the potential for locally administered adjuvant immunotherapies to manipulate the tumour milieu such that the tumour can now become its own vaccine.

This raises the possibility of using HS treated tumour lysates to complement current treatment options for the management of solid malignancies like MM. Thermal treatment modalities such as radio frequency ablation (RFA), microwave ablation (MVA) and cryoablation have been used to treat solid malignancies, including lung cancers [29-31]. RFA induces hyperthermia within the tumour resulting in necrotic cell death and the release of endogenous adjuvants such as Hsp70 [32], which can then promote a primary anti-tumour response. Indeed, the combination of HSL pulsed autologous DC vaccine with RFA was recently shown to abrogate tumour recurrence [33], demonstrating the practical advantage of using HS treatment of autologous vaccines as an adjuvant therapy in combination with standard treatment modalities to target residual disease.

5. Conclusion

In conclusion, we have shown that heat shocking of autologous tumour cells is a simple and translatable approach to generate an immunogenic lysate vaccine with significant prophylactic and therapeutic effects when delivered intratumourally. Coupling intratumoural autologous heat shocked vaccines with conventional therapies such as chemotherapy and surgery may improve patient responses for otherwise refractive tumours such as malignant mesothelioma.

6. Acknowledgements

The authors have no conflict of interest to report. This work was supported by the Dust Diseases Board, NSW, Australia. A.J. Currie was kindly supported by the Raine Medical Research Foundation, Western Australia.

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