T cell immunity required for tumor eradication following combination therapy.

<p>Mice were depleted of specific T cell subsets by systemic administration of antibodies against CD4, CD8 and NK cells and subjected to combined Id2kdN2a cells and α-CTLA-4 antibody treatment strategy (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129237#pone.0129237.g003" target="_blank">Fig 3A</a>) (<b>A</b>) <i>In vivo</i> depletion of CD4+, CD8+ and NK+ cells (n = 5 for all groups) shows that the therapeutic effect of the combined treatment was completely abrogated by CD8+ cell depletion. Mice lacking NK cells were for the most part able to reject their tumors following therapy. CD4+ T cell depletion initially appeared to have minimal adverse effects on the therapeutic vaccine strategy, but after 4 weeks all mice in the CD4 depletion group developed delayed tumors. (<b>B</b>) All CD8+ T cell depleted mice developed tumors at the site of Id2kdN2a cell vaccination as well. (<b>C</b>) Cell infiltrates from the tumors of CD4 depleted mice were stained with anti-CD45-FITC, CD4-APC and FoxP3-PerCPCy5.5 antibodies to identify Treg cells; there was no evidence of Treg cell infiltrate in the late developing tumors from CD4 depleted mice. Purified Treg cells were used as positive control.</p

Id2 knock down attenuates tumorigenicity and induces host immunity.

<p>(<b>A</b>) Sixty percent of mice (n = 9/15) challenged (right leg) with Id2 knock down Neuro2a (Id2kdN2a) cells rejected tumor and survived tumor-free, whereas all mice challenged with either wild type Neuro2a (wtN2a, n = 20) or scrambled shRNA lentivirus transfected Neuro2a (sc-shRNA-N2a, n = 5)) cells died from tumor burden. (<b>B</b>) Tumor free survivors from (A) were re-challenged with wtN2a cells into their left leg 6 weeks after they cleared the tumor and 8 out of 9 mice were protected from tumor growth. (<b>C</b>) SCID and nude mice grew tumors aggressively following inoculation with Id2kdN2a cells. (<b>D</b>) Following inoculation of wtN2a cells in the right leg, Id2kdN2a (n = 10) and wtN2a (n = 5) cells were vaccinated into the left leg of mice, 3 and 5 days later respectively. The wild type tumor growth on the right leg was delayed in Id2kdN2a vaccinated mice when compared to control unvaccinated mice or wtN2a vaccinated mice.</p

Tumor clearance and effect of irradiation on tumor cell immunogenicity.

<p>(<b>A</b>, <b>B</b>) Representative mouse from untreated and combination therapy group showing growing and shrinking tumor respectively. All bio-luminescent images were analyzed under the same scale.(<b>C</b>, <b>D</b>) Irradiated Id2 knock down Neuro2a (IR-Id2kdN2a, n = 5) and wild type Neuro2a (IR-wtN2a, n = 5) were compared as whole tumor cell vaccine antigen source in combination with α-CTLA-4 antibody against AgN2a (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129237#pone.0129237.g003" target="_blank">Fig 3A</a>). IR-Id2kdN2a vaccine was able to eradicate 60% of tumors in comparison to IR-wtN2a cells which had no effect on growth of the AgN2a tumors. All mice in (C) were sacrificed on day 20 due to large tumor burden. The parenthesis indicates number of mice that survived tumor free.</p

Combination of Id2kd N2a and α-CTLA4 antibody as a therapeutic vaccine.

<p>(<b>A</b>) Schematic diagram of the therapeutic vaccine strategy. Two established tumor models, namely Neuro2a (wtN2a) and AgN2a were tested, where mice challenged with either wtN2a or AgN2a cells were subjected to a combination immunotherapy with Id2kdN2a and α-CTLA4 antibody starting at day 6 after inoculation. Neuroblastoma tumors are normally visible (5mm in diameter) in AJ mice by day 6. Tumor growth curves in individual mice of wtN2a (<b>B</b>) and AgN2a (<b>C</b>) cells show that the Id2kd tumor cell vaccination combined with immune-modulation cures mice with established tumor. Parentheses indicate the number of mice that survived tumor free.</p

TIL in human neuroblastoma samples.

<p>Five freshly harvested human neuroblastoma specimens were analyzed for TIL; two of the tumors were from patients with Opsoclonus/myoclonus syndrome (OMS). (<b>A</b>) Representative flow cytometry plots showing CD4+ and CD8+ T-cell population in the neuroblastoma tumor lymphocytes. (<b>B</b>) Graphical representation of (A) indicating a remarkable infiltration of CD8+ T-cells in the two tumors associated with OMS (NB-OMS) as opposed to the other three neuroblastoma (NB) tumors that had minimal TIL.</p

Enhanced <i>in vivo</i> immune response mediates tumor clearance.

<p>(<b>A</b>) Representative flow cytometry plots showingCD4+ and CD8+ T-cells in CD45+ splenocytes of naïve (n = 5), tumor bearing (n = 3) and tumor free (n = 5) mice. (<b>B</b>) Graphical representation of (A) indicating significant (** p = 0.013) increase in CD8+ cells in the spleens of mice cleared of tumor. (<b>C</b>) Splenocytes of mice that cleared tumor had enhanced IFNγ secretion following stimulation with CD3 (* p<0.02),wtN2a or Id2kdN2a cells (*** p<0.0001). (<b>D</b>) Remarkable increase in CD45+ cells detected in the shrinking tumor.(<b>E</b>, <b>F</b>) Tumor infiltrating lymphocytes (TIL) following vaccination were quantified and a massive infiltration of CD8+ T-cells was detected in the shrinking tumors (n = 5) as opposed to the growing tumors (* p<0.02). (<b>G</b>) Chromium⁵¹ release assay exhibited potent cytotoxic activity of CD8+ TIL from shrinking tumor (n = 3); whereas the TIL isolated from growing tumors (n = 4) show no activity at all. Data presented as mean ± S.D.</p

Effect of Id2 down regulation on human neuroblastoma cell lines.

<p>(<b>A</b>) BrdU incorporation assay demonstrated that transfection of AD phenotype of SK-N-SH and IMR-32 cells with Id2-siRNA reduced the rate of proliferation. The apoptotic cells were excluded by gating out the sub-2n cells. (<b>B</b>) AnnexinV staining revealed increased apoptosis in AD phenotype of SK-N-SH and IMR-32 cells following transfection with Id2-siRNA. The percent apoptotic cells in the graphs represent the sum of early and late apoptosis. (<b>C</b>) Representative bands from Western blot analysis revealed over activation of Akt, Erk and Smad pathways and overexpression of Integrin β1 protein in both SK-N-SH and IMR-32 cells after Id2 down regulation. Data points represent mean ± S.D. (n = 4–6). *p<0.004 by Student’s t-test. Control or C: AD cells; siR or siRNA: AD cells transfected with siRNA against Id2; cR or cRNA: AD cells transfected with nonsense siRNA control.</p

Treatment targeting AD and AI phenotypes as well as adaptive plasticity delayed mouse tumor growth.

<p>(<b>A</b>) A graphical representation of the gross tumor presence demonstrates that the combined treatment with doxorubicin, metformin, sorafenib and LY2109761 cured 50% of mice with microscopic tumors. All treatments in (A) were started one day after neuro2a inoculation (n = 10–15). (<b>B</b>) Tumor volume curve during treatment of established tumors with a combination of doxorubicin, metformin, sorafenib and LY2109761 showed remarkably suppressed tumor growth (n = 10). All mice in the control group were sacrificed by day 11 after the tumors reached 20 mm in diameter. The drug treatment in (B) was started after 5 mm tumor was visible in mice. Dox: doxorubicin, met: metformin, soraf: sorafenib, LY: LY2109761.</p

A model depicting the role of Id2 in Neuro2a phenotypic transition.

<p>We show that the AI cells lose their proliferative potential due to loss of n-myc and Id2 expression when conditions induce loss of cell-matrix anchorage or serum starvation. The mechanism by which this occurs is through competitive binding of retinoblastoma (Rb) and TGFβ. Diminished Id2 enables Rb binding to E2F, thus blocking progression into the S-phase of cell cycling (free E2F induces S-phase genes) and inhibiting proliferation. Subsequently the cells either undergo apoptosis or develop resistance to anoikis. Concurrently, inhibiting Id2 enables TGFβ to activate the pathways of anoikis resistance allowing the cells to adapt to unfavorable conditions.</p

Id2 overexpression increases cell proliferation in Neuro2a AI cells.

<p>(<b>A</b>) Expression of green fluorescent protein (GFP) is evident in AI cells transfected with IRES-GFP or Id2-IRES-GFP plasmid. (<b>B</b>) Id2 protein is expressed in AI cells after Id2-IRES-GFP transfection, whereas Id2 protein was absent in AI cells transfected with IRES-GFP. (<b>C</b>) Representative plots showed 77–85% transfection efficiency. (<b>D, E</b>) BrdU incorporation assay indicating increased rate of proliferation in AI cells after Id2 overexpression. The apoptotic cells were excluded by gating out the sub-2n cells. (<b>F</b>) Representative bands from Western blot analysis revealed that overexpression of Id2 reduced the activation of Erk, Akt and Smad pathways in AI cells. Data points represent mean ± S.D. (n = 4–6). *p<0.04 by Student’s t-test. C: AI cells, Ires: AI cells transfected with IRES-GFP, Id2: AI cells transfected with Id2-IRES-GFP.</p