A specific protocol to induce anti-tumor effect by alum-only therapy

In our attempts to observe alum as an adjuvant to boost anti-tumor response against a Balb/c hepatoma line H22 initially established from a lymphatic metastatic model35, we immunized H22 tumor-bearing mice with various isogenic tumor cell lysates in combination with alum to detect any potential therapeutic effect. We noticed an occasional reduction of tumor size in alum alone group in the absence of tumor antigen (tumor lysate). To establish a protocol to capture this effect more consistently, we set up a series of experiments to identify the optimal immunization schedule. A representative scheme is outlined in Fig. 1A. H22 hepatocarcinoma cells were first inoculated i.p. in Balb/c mice, the peritoneal lavage was collected and 105 cells from the lavage were injected s.c. into new recipients. 5, 7 or 10 days later, 250 μg of Al(OH)3 in 250 μl of PBS or PBS alone was injected i.p. into the inoculated mice, followed by the same treatment every three or four days for a total of 6 injections (Fig. 1A). In all schedules of alum treatment, those initiated 7 or 10 days post tumor inoculation showed tumor volume increase similar to the PBS control. Interestingly, the treatment initiated 5 days after (alum 5DPI) exhibited significant growth reduction, with the differences becoming greater over time (Fig. 1B). We then selected this schedule for more detailed analyses. In line with the initial finding, tumors removed from the alum 5DPI-treated mice 25 days after the inoculation were visibly smaller than the PBS control (Fig. 1C,D). This was accompanied by a statistically significant survival advantage as no mice in the PBS group remained at day 54, in contrast with a 50% survival rate in the alum 5DPI group (Fig. 1E). Although in vivo growth potential of H22 changed over time likely as a consequence of cell culture which resulted in slightly different growth rates from experiment to experiment, this outcome nonetheless suggests that alum injection alone, administered after hepatocarcinoma establishment, in comparison with PBS control leads to an unexpected suppression on tumor growth.

Figure 1 The alum alone treatment inhibits H22 tumor growth. Groups of female Balb/c mice (seven per group) were inoculated s.c. with 105 per mouse of H22 tumor cells on day 0. Tumor-bearing mice were injected i.p. with 0.25 mg/250ul Al(OH)3 or 250 ul PBS, twice weekly for 3 consecutive weeks. (A) A schematic representation of treatment protocol. (B) Line graph depicting the tumor volume in H22 tumor-bearing mice receiving different treatments over time. (C) Tumor volume changes in H22 tumor-bearing mice treated with Al(OH)3 or PBS starting on the 5th day. (D) Photos of tumors from mice sacrificed and solid tumors isolated on day 22. (E) Kaplan-Meier survival analysis of each group. Each value represents mean ± SEM. *p < 0.05, **p < 0.01 and ****p < 0.0001. Unmarked comparisons are not statistically significant. Full size image

Enhanced immune activation in alum 5DPI-treated tumor-bearing mice

To probe the immunological changes associated with the tumor suppression in Fig. 1, we followed cytokine profile in peritoneal lavage and tumor homogenate over time. Figure 2A shows that IL-1β and IL-6 were slightly elevated in the lavage of the alum 5DPI mice in comparison to the PBS control, although the absolute quantities were quite low. In contrast, amounts of IL-1β and TNFα were increased in the alum-treated groups (Fig. 2B), suggesting the treatment is associated with an enhanced inflammatory response.

Figure 2 Enhanced immune activation in alum 5DPI-treated tumor-bearing mice. Balb/c mice were inoculated s.c. with 105 H22 cells and randomly divided into two groups 5 days later. Mice were treated with Al(OH)3 as indicated. Peritoneal wash fluid and tumor interstitial fluid were collected 3 days after the 1st, 3rd and 6th alum injection. Levels of IL-1β, IL-6, TNF-α and IL-10 in peritonea (A) and tumor (B) were analyzed by ELISA. (C) Nude mice were inoculated with 105 H22 cells and treated with Al(OH)3 or PBS. Line graph depicting the tumor volume in H22 tumor-bearing nude mice receiving alum or control treatment. (D) Kaplan-Meier survival analysis of H22 tumor-bearing nude mice. Each value represents mean ± SEM. *p < 0.05, **p < 0.01. Unmarked comparisons are not statistically significant. Full size image

Alum was administered spatial-temporally away from the H22 tumor inoculation. By default, the presence of particulate or crystalline structures can activate innate immune responses, particularly as a consequence of interaction with phagocytes. Whether the alum-mediated tumor suppression also required the adaptive immunity was of great interest. To that end, we carried out the experiment of Fig. 1 in nude Balb/c mice. Figure 2C,D show that the protective benefit wrought out by alum was lost in the absence of cellular immunity, as assessed by tumor size or mortality.

CD8 + T cells are essential to alum-associated tumor suppression

The control over neoplastic pathogenesis by the cellular immunity is highly complex. While CD8+ T cells can exert their effect via cytolysis and apoptosis induction, CD4+ T cells are increasingly recognized as an important factor as well, modulating cytokine production and tumor microenvironment36, in addition to a subset of these cells, regulatory T cells, serving as a balancing factor. On day 6, 9, 13, 16, 20 and 23 after inoculation, we i.p. infused blocking antibodies against CD8, CD4 and neutrophil marker Ly6G into mice treated with alum 5DPI. Figure 3A shows that depletion of CD3+ or CD8+ T cells essentially eliminated the protective effect; while CD4+ T cell removal produced a marginal, statistically insignificant reduction in protection. Neutrophil depletion also resulted in an intermediate reversal of protection. To confirm the role of CD8+ T cells, lymphocytes from alum 5DPI-treated groups were harvested after three injections and stimulated with H22 tumor lysate. The expansion of CD8+ T cells was analyzed by FACS and the division index (ratio of expansion in the presence over in the absence of tumor lysate) was plotted in Fig. 3B. Clearly, CD8+ T lymphocytes in the alum-treated mice showed substantial expansion, in comparison with the PBS control. While overall the percentage did not change in the blood, even slightly decreased in LNs that drained the site of inoculation, large numbers of CD8+ T cells infiltrated the tumor in the alum group (Fig. 3C). In keeping with the lesser involvement of CD4+ T cells, no change was seen for CD4+ T cells at these locations (Fig. 3D). Therefore, alum-treated mice show enhanced H22 tumor-specific CD8+ T cell immunity, while CD4+ T cell response does not appear to be critical. We failed to detect significant NK cells in the tumor (near or below 1%). On the other hand, Treg cells appeared in higher number on day 15 in PBS control in comparison with alum treated group, suggesting that alum might suppress the increase of Treg cells in tumor growth environment (Supplemental Fig. 1).

Figure 3 CD8+ T cells are essential for alum-induced tumor suppression. (A) Balb/c mice were inoculated s.c. with H22 cells and treated with Al(OH)3 or PBS. Tumor-bearing mice were injected i.p. with 100 μg of anti-Ly6G, anti-mouse CD8 or rat IgG2a κ isotype control antibody in 200 μl PBS 1 day after each Al(OH)3 injection. The tumor volume was measured every 3 days. (B) Draining LNs were harvested from tumor-bearing mice in PBS- and Al(OH)3-treated groups 15 days after tumor inoculation. Single cell suspensions were prepared, labeled with cell proliferation dye eFluor 670 and stimulated with 10 μg/ml H22 cell lysis (protein) for 72 h and analyzed by FACS. Peripheral blood, spleen, draining LNs and tumor were harvested from tumor-bearing mice 3 days after the 1st, 3rd, 6th injection. The percentages of CD8+ (C) and CD4+ (D) T cells in total T cells or in total cells were analyzed by FACS. P values for D15 CD8+ and CD4+ cells in draining lymph nodes and CD8+ cells in tumor are 0.035, 0.045 and 0.047 respectively. Full size image

Substantial changes in gene regulation and chemokine production following alum treatment

To globally analyze how the alum treatment impacts H22 tumor, a gene expression profile of surgically recovered tumor 15 days after inoculation was studied by microarray genechip analysis. After alum treatment there were 364 genes differentially expressed (Supplemental Fig. 2). Among these genes, 20 were up-regulated. All these genes can be divided into four categories: 1. suppression of tumorigenicity, such as St14 (Suppression of Tumorigenicity 14)37; 2. immune regulation, such as Wif1 (Wnt Inhibitory Factor 1)38; 3. apoptosis induction and proliferation inhibition, such as Dapk2 (Death-Associated Protein Kinase 2)39, Gas-1(Growth Arrest Specific 1)40; 4. anti-microbial infections, such as Ear2 (Eosinophil-Associated Ribonuclease 2), 10, 12 and 4B41,42 (Fig. 4A). Although the immediate impact of these gene regulations was not clear in alum-treated mice and factors such as chemokine changes were not detected (see below), this assay nonetheless showed a different profile suggestive of tumor suppression in those mice.

Figure 4 Changes in gene regulation and chemokine production following Al(OH)3 treatment. (A) Heat maps showing the hierarchical clustering of samples along genes in a fixed order for tumor sample in PBS- and Al(OH)3-treated groups. Tumors were harvested from tumor-bearing mice 15 days after the inoculation. Total RNA was extracted and quantitated as described in the methods. The comparison between two groups was carried out using three biological replicates. Shown are transcripts classified as tumor suppression-related and those expression levels significantly different between two groups. (B,C) Balb/c mice were inoculated with105 H22 cells, 5 days later, tumor-bearing mice were randomly divided into two groups. Peritoneal wash fluid and tumor interstitial fluid were collected from mice 3 days after the 1st, 3rd and 6th injection. CCL2, CXCL9 and CCL5 in peritonea (B) and tumor fluid (C) were analyzed by ELISA. Each value represents the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unmarked comparisons are not statistically significant. Full size image

We reasoned that infiltration of CD8+ T cells into the tumor was the result of chemotactic changes. Peritoneal lavages and lysates of recovered tumor were monitored for CCL2, CCL9 and CCL5 levels. Figure 4B shows that all these chemokines were present in greater amounts in alum treated group, at least on day 15 after the tumor inoculation. Interestingly, only CCL2 was found to be elevated in tumor lysate on day 15, in line with the ability of this chemokine to attract CD8+ T cells (Fig. 4C). Since it appeared quite late, this increase suggested a heightened monocytic infiltration concomitant with an increased CD8 response towards the end of treatment schedule.

Neutrophils enter the tumor

In Fig. 3A, Ly6G antibody treatment led to a perceivable reverse of alum’s anti-tumor effect. Unlike tumor antigen-specific CD8+ T cells, the association of neutrophils and tumor is more complex. While it is generally believed that these cells help shape a pro-growth microenvironment thus promoting tumor, more recent studies suggest that they in some cases also kill tumor cells43,44,45. In the alum-treated group, there was little change of overall neutrophil numbers in the blood, however, they were elevated in the tumor, suggesting that alum is conducive to a pro-inflammatory environment (Fig. 5A). To see if the recruited cells were more efficient in inducing tumor cell death, neutrophils recovered from the blood were used in a co-culture assay with H22 cells. Clearly, neutrophils from the alum-treated mice were much more efficient in mediating Annexin V turnover on the target cells than ones from the PBS group (Fig. 5B), in line with the reduced tumor suppression when Ly6G antibody was used to deplete this population.

Figure 5 Neutrophils in tumor. (A) Mice with established tumors were injected i.p. with Al(OH)3 or PBS. Peripheral blood, spleen, draining LNs and tumor were harvested from tumor-bearing mice on day 3 after the 1st, 3rd and 6th injections. The percentage of neutrophils in myeloid-derived cells or total cells of spleen, draining LNs and tumor were detected by FACS. (B) Neutrophils were purified from tumor-bearing mice in different groups and cocultured with eFluor 450-labeled H22 cells at ratio of 20:1. 12 hour later, tumor cells were gated and their apoptosis was evaluated (right chart). (C) The percentages of tumor cells killed were calculated. Each value represents the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unmarked comparisons are not statistically significant. Full size image

No adverse tissue damage following alum injection

In the absence of co-administered antigen, alum presumably exerts it anti-tumor effect by nonspecifically upregulating antigen presentation, to activate antigen-specific T cells. Without targeting specific antigens, it is possible that immune responses may be induced against a plethora of epitopes derived from tumor antigens, including those normally present on regular host cells. This could lead to autoimmunity. On the other hand, since in our protocol alum was administered repeatedly, its ability to provoke innate immunity could induce unintended tissue damage. Previous figures show that the overall immune cell numbers or cytokine levels were not drastically changed in the blood and the spleen, suggesting the lack of systemic damage. Spleen, kidney and lung sections were prepared following the full course of alum 5DPI treatment. H&E staining did not show any gross difference between samples from the PBS- and the alum-treated mice (Fig. 6A–C), confirming that repeated alum treatment is likely a safe protocol.