Neoadjuvant anti-tumor vaccination prior to surgery enhances survival

All reagents were purchased from Sigma Aldrich (Castle Hill, NSW. Australia) unless stated otherwise. Female BALB/C (H-2K^d) aged between 6 and 8 weeks were obtained from the Animal Resources Centre (Murdoch, Western Australia). All mice were maintained under standard specific pathogen free (SPF) housing conditions and all animal experiments were carried out according to protocols approved by the University of Western Australia Animal Ethics Committee.

Mice were inoculated subcutaneously (s.c.) with 5×10⁵ AB1-HA cells in a total volume of 100 μl PBS on the right hand flank unless otherwise stated. For all experiments mice were randomised into groups of 5 once tumors were established (approximately 50–70 mm²). Mice were culled when tumors reached the maximum allowable size of 100 mm² as per UWA AEC approvals. All surgical resections were performed under general anaesthetic using inhalant isoflurane. As a control for the surgical process sham surgery was performed by making incisions into the tumor mass and re-suturing, without debulking. Debulking surgery was performed via elliptic incisions, centred over the s.c. tumors. Skin flaps were elevated to expose adherent tumors. Once tumors were dissected clear of adjacent fascia, 75% of the tumor mass was removed, with preservation of the tumor pedicles to ensure blood supply (as previously described [19]). Wounds were closed using 4–0 vicryl (polyglactin 910, Ethicon, Australia) continuous sutures. Mice received 0.1 mg/kg buprenorphine intraperitoneally (i.p.) in the recovery phase for postoperative analgesia as required.

The murine mesothelioma cell line (AB1) was generated by injecting crocidolite asbestos i.p. into BALB/C mice as previously described [20] and subsequently transfected with the PR8 influenza virus haemagglutinin (HA) gene to produce AB1-HA [21].

Mouse adapted Influenza virus (PR/8/34/H1N1) was a kind gift from Dr Peter Henry (UWA School of Pharmacology, Perth Western Australia). PR8 virus was propagated in the allantoic fluid of 9-day old embryonated chicken eggs (Altona Hatchery, Forrestfield, Australia) at 37°C for 3 days and harvested as previously described [22], stored in single use aliquots at −80°C and diluted 1:100 to 1:400 in sterile PBS prior to intranasal vaccination of mice.

Generation of recombinant Modified Vaccinia Ankara expressing Influenza HA antigen. PR8 Influenza HA from the AB1-HA cell line was cloned via RT-PCR into pCR4 cloning vector using the pZeroBlunt-TOPO cloning kit. PCR primers were designed to introduce a Pme I restriction site and add a 6HIS-3xSTOP-Asc I sequence to the 5′ and 3′ end of the HA cds respectively. The Pme I/Asc I flanked HA-6HIS insert from pCR4-HA was then cloned into the modified Vaccinia Ankara (MVA) shuttle vector pZWIGR3 [23] to produce pZWIGR3-HA. To generate recombinant MVA (rMVA) expressing HA, pZWIGR3-HA was transfected into wild type MVA (wtMVA) infected BHK-21 cells and rMVA-HA positive cells purified via sequential rounds of plaque purification based on expression of the fluorescent reporter Venus. The purity of rMVA-HA stocks was confirmed by the absence of wtMVA and presence of HA via PCR prior to expansion and ultra-purification of rMVA-HA stocks for experimental use. All rMVA-HA vaccinations were via i.p. injection in a total volume of 100 μl PBS containing 5×10⁵ plaque forming units of virus.

Flow cytometry was performed using a BD Canto II. All antibodies are anti-mouse unless otherwise stated. Flow cytometry: αCD3-PE-Cy7 (clone 145-2C11), αCD4-PerCP-Cy5.5 (clone GK1.5), αCD8-FITC (clone 53-6.7) and αCD278(ICOS)-APC (clone C398.4A) all BioLegend, USA; αCD8-APC-ef780 (clone 53–6.7), αFoxP3-FITC (clone FJK-16 s), αIFNγ-APC (clone XMG1.2), αCD44-PerCP-Cy5.5 (clone IM7), αCCR7-PE (clone 4B12) and αCD62L-APC (Clone MEL-14) all eBioscience, USA; αKi67-PE (clone B56) (BD Biosciences, USA) and HA-Dextramer-APC (Immudex USA, LLC. Virginia USA). For depletion experiments, purified αCD4 (GK1.5) and αCD8 (YTS.169) antibodies were obtained from Absolutions Pty Ltd (Western Australian Institute for Medical Research, Perth, Western Australia). Antibodies were administered by intravenous (i.v.) injection at a dose of 150 μg per mouse on day 1 and then 100 μg i.p. per mouse every third day as indicated. T cell subset depletion was confirmed by flow cytometry on peripheral blood samples.

We have previously demonstrated that partial, but not complete debulking surgery promotes protective anti-tumour immunity when combined with adjuvant immunotherapy [7]. This is despite complete resection preventing any relapse of residual tumor growth [19]. We have continued to refine our model of tumor debulking by investigating the effect of removing different amounts of tumor on the rate of residual tumor outgrowth and overall survival (Khong et al., manuscript in preparation) When 75% of the tumor was debulked we observed that the outgrowth of the residual tumor was relatively slow, while in contrast, removing 50% or less of the tumor had no effect on tumor outgrowth. We therefore chose a 75% debulk model for this study as it better represents the many clinical scenarios where debulking surgery is the realistic goral rather than complete resection.

To determine whether the addition of PR8 prime and rMVA-HA boost (P/B) vaccination directed against the HA-neo tumour antigen (as describe above) could improve the survival benefit associated with debulking surgery, groups of AB1-HA tumor bearing mice were treated with either sham surgery (tumor incised, but not debulked), 75% debulking surgery, prime boost vaccination or a combination of neoadjuvant vaccination and surgery (Figure 1A). A significant delay in tumor growth (p < 0.001) was observed for surgery combined with vaccination group relative to other treatment groups. (Figure 1B). Groups receiving vaccination (alone or in combination with surgery) had noticeably smaller tumors on the day of surgery and a significantly higher proportion of interferon gamma (IFN-γ) expressing tumor-specific CD8 T cells when compared to non-vaccinated groups one week after vaccination (Figure 1D). However, despite all vaccinated groups demonstrating the presence of a functional tumour-specific CD8 T cell immune response, only the combination of neoadjuvant vaccination and 75% debulking surgery was associated with significantly delayed tumor growth and increased significant survival benefit (p < 0.001) relative to surgery or vaccination alone (Figure 1B).

We hypothesised that the delay in tumor growth in vaccinated mice resulted from a vaccine induced anti-tumor immune response. To test this, we depleted two known key anti-tumour immune effector cell types, CD4 and CD8 T lymphocytes, during vaccination and compared the overall survival of each group (Figure 2A). Consistent with our earlier experiments, we observed a delay in tumor growth that was associated with a significant survival benefit (p < 0.01) when neoadjuvant vaccination was combined with 75% debulking surgery compared to surgery alone; although this was not sufficient to prevent tumor outgrowth. Depletion of CD8 T cells, either alone or in combination with CD4 T cells, completely abrogated any vaccine induced survival benefit, reducing the survival of CD8-depleted groups to that of the surgery only group (Figure 2A), indicating that CD8 T cells are essential for an effective anti-tumor immune response. Similarly, CD4 depletion during vaccination prevented any vaccine induce delay in tumor growth prior to surgery. However, in contrast to the combined surgery and vaccine group, in which all mice eventually succumb to tumor, we observed complete tumor eradication in 60% of CD4 depleted mice following debulking surgery (Figure 2A). Further experiments confirmed that CD4 depletion either prior to surgery, or during vaccination was sufficient to eradicate tumor in 50% (5/10) of treated animals and this could be increased to 70% (7/10) when vaccination was combined with surgery (Figure 2B), demonstrating that vaccination with CD4 depletion significantly enhances survival compared to surgery alone. In both sets of experiments all mice survived tumor-free for longer than 60 days post-surgery. In addition, they resisted rechallenge with the parental tumor cell-line that does not express HA (black arrow, Figure 2A & B), indicating that the vaccine-induced immune response was against shared tumor antigens on the AB1 mesothelioma cells and not solely against the transfected HA antigen.

Having demonstrated that CD8 T cells were essential for effective anti-tumor immunity, we next assessed the immunological effect of CD4 depletion on CD8 T cells. Peripheral blood taken from mice during CD4 T cell depletion (days 7, 10, 15 and 22, shown in Figure 2B) was analysed by polychromatic flow cytometry to assess the proportion and activation status (ICOS expression) of T cell subsets. The proportion of CD4 and CD8 (as a percentage of total CD3+ lymphocytes) and FoxP3 + CD4+ regulatory T cells (Treg, as a proportion of total CD4+ T cells) was as expected, the same in all groups at baseline (day 7) and remained unchanged throughout the experiment in all non-CD4 depleted groups (Figure 3). In contrast, the proportion of CD4+ T cells and Treg were significantly depleted upon anti-CD4 mAb treatment (p < 0.0001), correlating with a significant increase in the proportion and activation status of CD8 T cells (Figure 3C-D, p < 0.0001). Sustained CD8 T cell activation was observed in CD4 depleted groups, coinciding with tumor regression (days 22–35) and ultimately long term survival (Figure 2B). CD4 T cell depletion was transient and had returned to baseline levels by day 81 when surviving mice were rechallenged with parental tumour (data not shown). Again, all surviving mice resisted rechallenge and remained tumor free until completion of the experiment (day 154). Establishment of immunological memory was consistent with the finding of a significant increase in CD44+ CD62L- effector memory T cells (T_EM) in the spleen and lymph nodes of CD4 depleted groups at day 140 relative to naïve bearing controls (Figure 4).