Sexual dimorphism in the antitumor immune responses elicited by the combination of fasting and chemotherapy

IF 20.1 1区 医学 Q1 ONCOLOGY Cancer Communications Pub Date : 2024-03-21 DOI:10.1002/cac2.12535
Andrés Pastor-Fernández, Manuel Montero Gómez de las Heras, Jose Ignacio Escrig-Larena, Marta Barradas, Cristina Pantoja, Adrian Plaza, Jose Luis Lopez-Aceituno, Esther Durán, Alejo Efeyan, Maria Mittelbrunn, Lola Martinez, Pablo Jose Fernandez-Marcos
{"title":"Sexual dimorphism in the antitumor immune responses elicited by the combination of fasting and chemotherapy","authors":"Andrés Pastor-Fernández,&nbsp;Manuel Montero Gómez de las Heras,&nbsp;Jose Ignacio Escrig-Larena,&nbsp;Marta Barradas,&nbsp;Cristina Pantoja,&nbsp;Adrian Plaza,&nbsp;Jose Luis Lopez-Aceituno,&nbsp;Esther Durán,&nbsp;Alejo Efeyan,&nbsp;Maria Mittelbrunn,&nbsp;Lola Martinez,&nbsp;Pablo Jose Fernandez-Marcos","doi":"10.1002/cac2.12535","DOIUrl":null,"url":null,"abstract":"<p>Fasting reduces chemotherapy toxicity [<span>1</span>], enhances immunogenic tumor cell death [<span>2, 3</span>] and increases CD8<sup>+</sup> T cell infiltration in tumors, particularly when combined with chemotherapy [<span>2, 3</span>] or immunotherapy [<span>4</span>]. Moreover, fasting exhibits a sexual dimorphism in the immune system [<span>5</span>].</p><p>The aim of our study was to elucidate the role of sex in the beneficial anti-tumoral effects of combining fasting and chemotherapy. For this, we inoculated B16-F10-derived melanoma allografts into immunocompetent male and female mice. Three days later, the mice were divided into: (1) not treated; (2) two cycles of 48-hour fasting; (3) two cycles of 10 mg/kg doxorubicin; (4) two cycles of doxorubicin and fasting for 24 hours before and 24 hours after doxorubicin inoculation (“combination treatment” or “CT”). The study methods are shown in the Supplementary Material file. Doxorubicin and fasting alone reduced tumor growth in both sexes with the same efficacy, and CT amplified this effect only in males (Figure 1A and Supplementary Figure S1A-C). Male mice bearing YUMM1.7 melanoma-derived tumors responded to fasting and doxorubicin, but females were insensitive to any of them (Figure 1B and Supplementary Figure S2A-C). Oxaliplatin did not affect B16-F10 tumor growth (Supplementary Figure S3A-D). Fasting reduced serum levels of testosterone only in males ([<span>6, 7</span>] and Supplementary Figure S4A). To explore the role of testosterone, we castrated males or implanted testosterone pellets in females. CT lost efficacy in castrated males and became efficient in females with testosterone pellets (Figure 1C and Supplementary Figure S4B-E). Next, we inoculated mice with MC38 colon carcinoma cells [<span>8</span>]. Oxaliplatin or fasting reduced tumor growth, and CT amplified this effect in both sexes (Figure 1D and Supplementary Figure S5A-C). Our findings indicate that sexual dimorphism occurs in different tumor types, is dependent on tumor and chemotherapy type, and testosterone is a key player in this sexual dimorphism.</p><p>To study the immune response in B16-F10 allografts treated with doxorubicin and/or fasting (Supplementary Figure S6A-D), we analyzed relevant immune cell types in inguinal lymph nodes (LN), peripheral blood (B) and tumors (T) (Supplementary Table S1-S4). CT increased stage II Natural Killer (NK) and Natural Killer T (NKT) cells in B16-F10 tumors only in males (Figure 1E-F and Supplementary Figure S6E-H). Females on CT had more exhausted CD8<sup>+</sup> T cells in their tumors (Figure 1G and Supplementary Figure S6I-K). Tumor-infiltrated CD8<sup>+</sup> T cells were functionally more active in CT in males (Supplementary Figure S7A-C), while serum TNFα did not change (Supplementary Figure S7D-E). Immunoablation of CD8 cells in male mice tended to reduce CT efficacy, which still improved the antitumor response (Supplementary Figure S8A-F and Supplementary Table S5), indicating that immune cell populations other than CD8 cells were also involved in this response. Evolution with treatment and sex of all other analyzed populations is shown in Supplementary Figure S9A-L and S10A-J. The transcription of many cytokines and chemokines was strongly upregulated in CT only in males (Supplementary Figure S11A-B). Finally, we treated male and female Hsd:Athymic Nude-<i>Foxn1<sup>nu</sup></i> mice lacking T lymphocytes with the same protocol. Fasting alone did not reduce tumor progression; single chemotherapy reduced tumor growth, and CT enhanced chemotherapy efficacy mostly in male mice (Supplementary Figure S12A-H). These results suggest that the beneficial effects of CT are dependent on the cellular immune system, particularly on NK and NKT cells. We then performed a high-dimensional analysis of the immune populations in the tumors using 17 surface markers representing relevant immune populations (Supplementary Table S6). After a dimensional reduction and unsupervised clustering, we obtained 13 immune clusters (Figure 1H and Supplementary Figure S13A) and quantified the differential presence of these immune cell types between experimental groups (Supplementary Figure S13B). Cluster 1 (M2 macrophages) was increased in chemotherapy and CT only in males (Supplementary Figure S13C), coinciding with Supplementary Figure S10J. Clusters 4 and 13, expressing markers of exhaustion (TIM-3 and PD-1), tended to be higher in females on CT (Figure 1I and Supplementary Figure S13D), confirming Figure 1G. Cluster 7 (stage I NKT) was significantly increased in CT compared with chemotherapy alone in both sexes (Supplementary Figure S13E). Next, we focused on CD8<sup>+</sup> T cells (Supplementary Figure S14A-B). Cluster 14 (exhausted central memory/effector CD8 cells) was increased following CT in both sexes (Supplementary Figure S14C-D). Clusters 18 and 20 (regulatory CD8 T cells [<span>9</span>]) tended to be decreased only in males with chemotherapy alone (Supplementary Figure S14C and E). These findings stress the differential response of CD8 cells between both sexes to chemotherapy and CTs. We also checked for sub-clusters within the NK1.1<sup>+</sup> cells and did not find informative sub-clusters (Supplementary Figures S14F-G).</p><p>We then analyzed the immune populations in mice bearing MC38 colon carcinoma cells (Figure 1D). CT increased total intratumoral effector and exhausted CD8<sup>+</sup> cells (Figure 1J and Supplementary Figure S15A-D). CD8 tumor infiltration was more active in fasting, chemotherapy and in CT than in the untreated mice (Supplementary Figure S15E-F). CT also increased intratumoral CD4 Th1 cells, with anti-tumoral properties (Figure 1K and Supplementary Figure S16A). Total intratumoral macrophages, with pro-tumoral properties, were decreased with CT (Figure 1L and Supplementary Figure S16B). The evolution of all other analyzed populations in both sexes is shown in Supplementary Figures S17-19, where populations in females behaved very similarly to males. Tumor cytokine and chemokine transcription did not significantly change with treatment or sex (Supplementary Figure S20A-B). High-dimensional analysis of the intratumoral immune populations using 19 surface markers (Supplementary Table S7), followed by a dimensional reduction and unsupervised clustering, generated 15 clusters (Figure 1M and Supplementary Figure S21A). Cluster 1 (M-MDSC) was reduced with chemotherapy and CT (Supplementary Figure S21B-C); Cluster 2 (PMN-MDSC) was increased in CT, especially in males (Supplementary Figure S21B and D); and Cluster 13 (tumor-associated macrophages) was reduced in both treatments and sexes (Figure 1N and Supplementary Figure S21B). The evolution of these three populations fit with our previous results (Figure 1L and Supplementary Figure S18I-L and S19C).</p><p>We analyzed tumor-draining lymph nodes and blood for a physiological understanding of the immune response. CT increased lymph node MDSC only in females bearing B16-F10 (Supplementary Figure S10B), partly explaining the failure of females to respond to fasting, and this change was not reflected in the tumors (Supplementary Figure S10A). CT increased lymph node-relevant central memory and naïve CD8 cells in the B16-F10 model in both sexes (Supplementary Figure S9C-D), and this was not reflected in blood or tumors. NK and NKT cells were important in the B16-F10 model: in males, CT decreased NK cells in the blood and increased them in the tumor (Supplementary Figure S9E), while stage II NKT cells were only increased in the tumor (Figure 1F). Comparing both tumor models, doxorubicin reduced total immune cells in blood and lymph nodes but oxaliplatin did not (Supplementary Figures S9A and S17A). These results indicate different global responses of the immune system between tumor types and that changes in blood or lymph nodes did not reflect those observed in tumors, in contrast to previous reports [<span>10</span>].</p><p>Our work confirms the beneficial effects of combining fasting with chemotherapy (doxorubicin and oxaliplatin) in mice. We observe for the first time a sexual dimorphism in this process with relevant clinical implications. Finally, we show that different tumor models show distinct immune responses, and therefore, the chemotherapy-enhancing ability of fasting may not depend on specific immune populations (Figure 1O).</p><p><b>Andrés Pastor-Fernández</b>: conceptualization; experimental design and performance; writing manuscript. <b>Manuel Montero Gómez de las Heras and Jose Ignacio Escrig-Larena</b>: high-dimensional immune methodology; formal analysis and investigation. <b>Marta Barradas</b>: immunofluorescence study design and analysis. <b>Cristina Pantoja</b>: project administration; data acquisition and curation, and mouse sample processing. <b>Adrian Plaza</b>: mouse sample processing; investigation and data interpretation. <b>Jose Luis Lopez-Aceituno</b>: mouse sample processing and technique optimization. <b>Esther Durán</b>: sample processing for immunofluorescence studies. <b>Alejo Efeyan</b>: support with immunofluorescence experiments. <b>Maria Mittelbrunn</b>: support with high-dimensional cytometry experiments. <b>Lola Martinez</b>: support with classical cytometry experiments; Pablo Jose Fernandez-Marcos: conceptualization; experimental design; funding and manuscript writing.</p><p>The authors have declared that no conflict of interest exists.</p><p>Andrés Pastor Fernández was a recipient of a predoctoral fellowship from the Spanish Association Against Cancer – AECC (PRDMA18011PAST). Cristina Pantoja and Marta Barradas were funded by the Madrid Institute for Advanced Studies (IMDEA) Food. Adrián Plaza was funded by the AECC (SIRTBIO-LABAE18008FERN). Jose Luis Lopez-Aceituno was funded by the Spanish Ministry of Science and Innovation (MICINN) (PTA2017-14689-I). Pablo Jose Fernandez-Marcos was funded by a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335 /AEI/10.13039/501100011033). Work at the laboratory of Pablo Jose Fernández-Marcos was funded by the AECC (SIRTBIO- LABAE18008FERN) and the RETOS Program projects from the MICINN (SAF2017-85766-R/AEI/10.13039/501100011033 and PID2020-114077RB-I00/AEI/10.13039/501100011033). Work in Lola Martínez flow cytometry unit was funded by the CNIO. Manuel Montero Gómez de las Heras and Jose Ignacio Escrig-Larena were supported by FPU grants (FPU19/02576 and FPU20/04066, respectively) from the Spanish Ministry of Science, Innovation and Universities. Work in the laboratory of Maria Mittelbrunn was supported by the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI19/855), the European Regional Development Fund (ERDF) and the European Commission through H2020-EU.1.1, European Research Council grant ERC-2016-StG 715322-EndoMitTalk, and the Y2020/BIO-6350 NutriSION-CM synergy grant from Comunidad de Madrid. Esther Durán was funded by the Centro de Estudios Universitarios (CEU) San Pablo University. Alejo Efeyan is an EMBO Young Investigator. Alejo Efeyan lab is supported by the Retos Projects Program of the Spanish Ministry of Science, Innovation and Universities, the Spanish State Research Agency (AEI/10.13039/501100011033), co-funded by the European Regional Development Fund (PID 2019-104012RB-I00), a FERO Grant for Research in Oncology, and La Caixa Foundation (HR21-00046).</p><p>All animal experiments were performed according to the protocols approved by the Spanish National Research Council (CSIC) Ethics Committee for Research and Animal Welfare in Spain and all the appropriate official entities (PROEX 148/18 and 249.3/20).</p><p>Not applicable.</p>","PeriodicalId":9495,"journal":{"name":"Cancer Communications","volume":null,"pages":null},"PeriodicalIF":20.1000,"publicationDate":"2024-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cac2.12535","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cancer Communications","FirstCategoryId":"3","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cac2.12535","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"ONCOLOGY","Score":null,"Total":0}
引用次数: 0

Abstract

Fasting reduces chemotherapy toxicity [1], enhances immunogenic tumor cell death [2, 3] and increases CD8+ T cell infiltration in tumors, particularly when combined with chemotherapy [2, 3] or immunotherapy [4]. Moreover, fasting exhibits a sexual dimorphism in the immune system [5].

The aim of our study was to elucidate the role of sex in the beneficial anti-tumoral effects of combining fasting and chemotherapy. For this, we inoculated B16-F10-derived melanoma allografts into immunocompetent male and female mice. Three days later, the mice were divided into: (1) not treated; (2) two cycles of 48-hour fasting; (3) two cycles of 10 mg/kg doxorubicin; (4) two cycles of doxorubicin and fasting for 24 hours before and 24 hours after doxorubicin inoculation (“combination treatment” or “CT”). The study methods are shown in the Supplementary Material file. Doxorubicin and fasting alone reduced tumor growth in both sexes with the same efficacy, and CT amplified this effect only in males (Figure 1A and Supplementary Figure S1A-C). Male mice bearing YUMM1.7 melanoma-derived tumors responded to fasting and doxorubicin, but females were insensitive to any of them (Figure 1B and Supplementary Figure S2A-C). Oxaliplatin did not affect B16-F10 tumor growth (Supplementary Figure S3A-D). Fasting reduced serum levels of testosterone only in males ([6, 7] and Supplementary Figure S4A). To explore the role of testosterone, we castrated males or implanted testosterone pellets in females. CT lost efficacy in castrated males and became efficient in females with testosterone pellets (Figure 1C and Supplementary Figure S4B-E). Next, we inoculated mice with MC38 colon carcinoma cells [8]. Oxaliplatin or fasting reduced tumor growth, and CT amplified this effect in both sexes (Figure 1D and Supplementary Figure S5A-C). Our findings indicate that sexual dimorphism occurs in different tumor types, is dependent on tumor and chemotherapy type, and testosterone is a key player in this sexual dimorphism.

To study the immune response in B16-F10 allografts treated with doxorubicin and/or fasting (Supplementary Figure S6A-D), we analyzed relevant immune cell types in inguinal lymph nodes (LN), peripheral blood (B) and tumors (T) (Supplementary Table S1-S4). CT increased stage II Natural Killer (NK) and Natural Killer T (NKT) cells in B16-F10 tumors only in males (Figure 1E-F and Supplementary Figure S6E-H). Females on CT had more exhausted CD8+ T cells in their tumors (Figure 1G and Supplementary Figure S6I-K). Tumor-infiltrated CD8+ T cells were functionally more active in CT in males (Supplementary Figure S7A-C), while serum TNFα did not change (Supplementary Figure S7D-E). Immunoablation of CD8 cells in male mice tended to reduce CT efficacy, which still improved the antitumor response (Supplementary Figure S8A-F and Supplementary Table S5), indicating that immune cell populations other than CD8 cells were also involved in this response. Evolution with treatment and sex of all other analyzed populations is shown in Supplementary Figure S9A-L and S10A-J. The transcription of many cytokines and chemokines was strongly upregulated in CT only in males (Supplementary Figure S11A-B). Finally, we treated male and female Hsd:Athymic Nude-Foxn1nu mice lacking T lymphocytes with the same protocol. Fasting alone did not reduce tumor progression; single chemotherapy reduced tumor growth, and CT enhanced chemotherapy efficacy mostly in male mice (Supplementary Figure S12A-H). These results suggest that the beneficial effects of CT are dependent on the cellular immune system, particularly on NK and NKT cells. We then performed a high-dimensional analysis of the immune populations in the tumors using 17 surface markers representing relevant immune populations (Supplementary Table S6). After a dimensional reduction and unsupervised clustering, we obtained 13 immune clusters (Figure 1H and Supplementary Figure S13A) and quantified the differential presence of these immune cell types between experimental groups (Supplementary Figure S13B). Cluster 1 (M2 macrophages) was increased in chemotherapy and CT only in males (Supplementary Figure S13C), coinciding with Supplementary Figure S10J. Clusters 4 and 13, expressing markers of exhaustion (TIM-3 and PD-1), tended to be higher in females on CT (Figure 1I and Supplementary Figure S13D), confirming Figure 1G. Cluster 7 (stage I NKT) was significantly increased in CT compared with chemotherapy alone in both sexes (Supplementary Figure S13E). Next, we focused on CD8+ T cells (Supplementary Figure S14A-B). Cluster 14 (exhausted central memory/effector CD8 cells) was increased following CT in both sexes (Supplementary Figure S14C-D). Clusters 18 and 20 (regulatory CD8 T cells [9]) tended to be decreased only in males with chemotherapy alone (Supplementary Figure S14C and E). These findings stress the differential response of CD8 cells between both sexes to chemotherapy and CTs. We also checked for sub-clusters within the NK1.1+ cells and did not find informative sub-clusters (Supplementary Figures S14F-G).

We then analyzed the immune populations in mice bearing MC38 colon carcinoma cells (Figure 1D). CT increased total intratumoral effector and exhausted CD8+ cells (Figure 1J and Supplementary Figure S15A-D). CD8 tumor infiltration was more active in fasting, chemotherapy and in CT than in the untreated mice (Supplementary Figure S15E-F). CT also increased intratumoral CD4 Th1 cells, with anti-tumoral properties (Figure 1K and Supplementary Figure S16A). Total intratumoral macrophages, with pro-tumoral properties, were decreased with CT (Figure 1L and Supplementary Figure S16B). The evolution of all other analyzed populations in both sexes is shown in Supplementary Figures S17-19, where populations in females behaved very similarly to males. Tumor cytokine and chemokine transcription did not significantly change with treatment or sex (Supplementary Figure S20A-B). High-dimensional analysis of the intratumoral immune populations using 19 surface markers (Supplementary Table S7), followed by a dimensional reduction and unsupervised clustering, generated 15 clusters (Figure 1M and Supplementary Figure S21A). Cluster 1 (M-MDSC) was reduced with chemotherapy and CT (Supplementary Figure S21B-C); Cluster 2 (PMN-MDSC) was increased in CT, especially in males (Supplementary Figure S21B and D); and Cluster 13 (tumor-associated macrophages) was reduced in both treatments and sexes (Figure 1N and Supplementary Figure S21B). The evolution of these three populations fit with our previous results (Figure 1L and Supplementary Figure S18I-L and S19C).

We analyzed tumor-draining lymph nodes and blood for a physiological understanding of the immune response. CT increased lymph node MDSC only in females bearing B16-F10 (Supplementary Figure S10B), partly explaining the failure of females to respond to fasting, and this change was not reflected in the tumors (Supplementary Figure S10A). CT increased lymph node-relevant central memory and naïve CD8 cells in the B16-F10 model in both sexes (Supplementary Figure S9C-D), and this was not reflected in blood or tumors. NK and NKT cells were important in the B16-F10 model: in males, CT decreased NK cells in the blood and increased them in the tumor (Supplementary Figure S9E), while stage II NKT cells were only increased in the tumor (Figure 1F). Comparing both tumor models, doxorubicin reduced total immune cells in blood and lymph nodes but oxaliplatin did not (Supplementary Figures S9A and S17A). These results indicate different global responses of the immune system between tumor types and that changes in blood or lymph nodes did not reflect those observed in tumors, in contrast to previous reports [10].

Our work confirms the beneficial effects of combining fasting with chemotherapy (doxorubicin and oxaliplatin) in mice. We observe for the first time a sexual dimorphism in this process with relevant clinical implications. Finally, we show that different tumor models show distinct immune responses, and therefore, the chemotherapy-enhancing ability of fasting may not depend on specific immune populations (Figure 1O).

Andrés Pastor-Fernández: conceptualization; experimental design and performance; writing manuscript. Manuel Montero Gómez de las Heras and Jose Ignacio Escrig-Larena: high-dimensional immune methodology; formal analysis and investigation. Marta Barradas: immunofluorescence study design and analysis. Cristina Pantoja: project administration; data acquisition and curation, and mouse sample processing. Adrian Plaza: mouse sample processing; investigation and data interpretation. Jose Luis Lopez-Aceituno: mouse sample processing and technique optimization. Esther Durán: sample processing for immunofluorescence studies. Alejo Efeyan: support with immunofluorescence experiments. Maria Mittelbrunn: support with high-dimensional cytometry experiments. Lola Martinez: support with classical cytometry experiments; Pablo Jose Fernandez-Marcos: conceptualization; experimental design; funding and manuscript writing.

The authors have declared that no conflict of interest exists.

Andrés Pastor Fernández was a recipient of a predoctoral fellowship from the Spanish Association Against Cancer – AECC (PRDMA18011PAST). Cristina Pantoja and Marta Barradas were funded by the Madrid Institute for Advanced Studies (IMDEA) Food. Adrián Plaza was funded by the AECC (SIRTBIO-LABAE18008FERN). Jose Luis Lopez-Aceituno was funded by the Spanish Ministry of Science and Innovation (MICINN) (PTA2017-14689-I). Pablo Jose Fernandez-Marcos was funded by a Ramon y Cajal Award from the Spanish Ministry of Science, Innovation and Universities (MICINN) (RYC-2017-22335 /AEI/10.13039/501100011033). Work at the laboratory of Pablo Jose Fernández-Marcos was funded by the AECC (SIRTBIO- LABAE18008FERN) and the RETOS Program projects from the MICINN (SAF2017-85766-R/AEI/10.13039/501100011033 and PID2020-114077RB-I00/AEI/10.13039/501100011033). Work in Lola Martínez flow cytometry unit was funded by the CNIO. Manuel Montero Gómez de las Heras and Jose Ignacio Escrig-Larena were supported by FPU grants (FPU19/02576 and FPU20/04066, respectively) from the Spanish Ministry of Science, Innovation and Universities. Work in the laboratory of Maria Mittelbrunn was supported by the Fondo de Investigación Sanitaria del Instituto de Salud Carlos III (PI19/855), the European Regional Development Fund (ERDF) and the European Commission through H2020-EU.1.1, European Research Council grant ERC-2016-StG 715322-EndoMitTalk, and the Y2020/BIO-6350 NutriSION-CM synergy grant from Comunidad de Madrid. Esther Durán was funded by the Centro de Estudios Universitarios (CEU) San Pablo University. Alejo Efeyan is an EMBO Young Investigator. Alejo Efeyan lab is supported by the Retos Projects Program of the Spanish Ministry of Science, Innovation and Universities, the Spanish State Research Agency (AEI/10.13039/501100011033), co-funded by the European Regional Development Fund (PID 2019-104012RB-I00), a FERO Grant for Research in Oncology, and La Caixa Foundation (HR21-00046).

All animal experiments were performed according to the protocols approved by the Spanish National Research Council (CSIC) Ethics Committee for Research and Animal Welfare in Spain and all the appropriate official entities (PROEX 148/18 and 249.3/20).

Not applicable.

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禁食和化疗联合疗法引起的抗肿瘤免疫反应的性别双态性。
禁食可减轻化疗毒性[1],增强免疫原性肿瘤细胞死亡[2, 3],增加CD8+ T细胞在肿瘤中的浸润,尤其是在与化疗[2, 3]或免疫疗法[4]联合使用时。我们的研究旨在阐明性别在禁食和化疗联合治疗的有益抗肿瘤效应中的作用。为此,我们将 B16-F10 黑色素瘤异种移植物接种到免疫功能正常的雌雄小鼠体内。三天后,小鼠被分为:(1)未接受治疗;(2)两个周期的 48 小时禁食;(3)两个周期的 10 毫克/千克多柔比星;(4)两个周期的多柔比星以及多柔比星接种前 24 小时和接种后 24 小时的禁食("联合治疗 "或 "CT")。研究方法见补充材料文件。单独使用多柔比星和禁食能以相同的疗效减少雌雄小鼠的肿瘤生长,而CT仅在雄性小鼠中扩大了这一效果(图1A和补充图S1A-C)。携带 YUMM1.7 黑色素瘤衍生肿瘤的雄性小鼠对禁食和多柔比星有反应,但雌性小鼠对任何一种药物都不敏感(图 1B 和补充图 S2A-C)。奥沙利铂不影响 B16-F10 肿瘤的生长(补充图 S3A-D)。空腹只降低了男性的血清睾酮水平([6, 7]和补充图 S4A)。为了探究睾酮的作用,我们对雄性动物进行了阉割,或在雌性动物体内植入了睾酮颗粒。CT在阉割的雄性小鼠中失去了效力,而在植入睾酮颗粒的雌性小鼠中变得有效(图 1C 和补充图 S4B-E)。接着,我们给小鼠接种了 MC38 结肠癌细胞[8]。奥沙利铂或禁食可减少肿瘤的生长,而CT可放大这种效应(图1D和补充图S5A-C)。为了研究多柔比星和/或禁食治疗的 B16-F10 异体移植物的免疫反应(补充图 S6A-D),我们分析了腹股沟淋巴结(LN)、外周血(B)和肿瘤(T)中的相关免疫细胞类型(补充表 S1-S4)。CT仅在男性中增加了B16-F10肿瘤中的II期自然杀伤细胞(NK)和自然杀伤T细胞(NKT)(图1E-F和补充图S6E-H)。服用 CT 的女性肿瘤中有更多衰竭的 CD8+ T 细胞(图 1G 和补充图 S6I-K)。肿瘤浸润的 CD8+ T 细胞在男性 CT 中功能更活跃(补充图 S7A-C),而血清 TNFα 没有变化(补充图 S7D-E)。雄性小鼠的 CD8 细胞免疫消融往往会降低 CT 的疗效,但仍能改善抗肿瘤反应(补充图 S8A-F 和补充表 S5),这表明 CD8 细胞以外的免疫细胞群也参与了这种反应。补充图 S9A-L 和 S10A-J 显示了所有其他分析细胞群随着治疗和性别的变化。许多细胞因子和趋化因子的转录仅在雄性 CT 中强烈上调(补充图 S11A-B)。最后,我们用相同的方案处理了缺乏 T 淋巴细胞的雌雄 Hsd:Athymic Nude-Foxn1nu 小鼠。单独禁食并不能减少肿瘤的进展;单一化疗能减少肿瘤的生长,而CT主要能增强雄性小鼠的化疗效果(附图S12A-H)。这些结果表明,CT 的有益作用依赖于细胞免疫系统,尤其是 NK 和 NKT 细胞。然后,我们利用代表相关免疫群体的 17 个表面标记物对肿瘤中的免疫群体进行了高维分析(补充表 S6)。经过降维和无监督聚类,我们得到了 13 个免疫聚类(图 1H 和补充图 S13A),并量化了这些免疫细胞类型在不同实验组之间的差异(补充图 S13B)。簇 1(M2 巨噬细胞)在化疗中增加,而仅在男性 CT 中增加(补充图 S13C),与补充图 S10J 一致。表达衰竭标志物(TIM-3 和 PD-1)的第 4 组和第 13 组在接受 CT 的女性中往往较高(图 1I 和补充图 S13D),证实了图 1G 的结果。与单纯化疗相比,CT 中的第 7 组(I 期 NKT)在男女患者中均显著增加(补充图 S13E)。接下来,我们重点研究了 CD8+ T 细胞(补充图 S14A-B)。第 14 组(衰竭的中枢记忆/效应 CD8 细胞)在男女患者 CT 后都有所增加(补充图 S14C-D)。第 18 群和第 20 群(调节性 CD8 T 细胞 [9])只有在男性单独接受化疗后才有减少的趋势(补充图 S14C 和 E)。这些发现强调了CD8细胞在两性之间对化疗和CT的不同反应。 我们还检查了 NK1.1+ 细胞内的亚群,但没有发现有信息的亚群(补充图 S14F-G)。我们随后分析了携带 MC38 结肠癌细胞的小鼠的免疫群体(图 1D)。CT 增加了瘤内效应细胞和衰竭 CD8+ 细胞的总数(图 1J 和补充图 S15A-D)。与未治疗的小鼠相比,禁食、化疗和 CT 中的 CD8 肿瘤浸润更活跃(补充图 S15E-F)。CT 还增加了具有抗肿瘤特性的瘤内 CD4 Th1 细胞(图 1K 和补充图 S16A)。具有促癌特性的瘤内巨噬细胞总数随着 CT 的使用而减少(图 1L 和补充图 S16B)。补充图 S17-19 显示了两性中所有其他分析种群的演变情况,其中女性种群的表现与男性非常相似。肿瘤细胞因子和趋化因子的转录没有随治疗或性别发生显著变化(补充图 S20A-B)。利用 19 个表面标记物(补充表 S7)对瘤内免疫群体进行高维分析,然后进行降维和无监督聚类,产生了 15 个聚类(图 1M 和补充图 S21A)。簇1(M-MDSC)在化疗和CT中减少(补充图S21B-C);簇2(PMN-MDSC)在CT中增加,尤其是在男性中(补充图S21B和D);簇13(肿瘤相关巨噬细胞)在两种治疗和性别中均减少(图1N和补充图S21B)。我们分析了肿瘤排出的淋巴结和血液,以从生理学角度了解免疫反应。CT只增加了B16-F10雌性淋巴结MDSC(补充图S10B),这部分解释了雌性淋巴结MDSC未能对禁食产生反应,而这种变化并没有反映在肿瘤中(补充图S10A)。在 B16-F10 模型中,CT 增加了淋巴结相关的中央记忆细胞和幼稚 CD8 细胞,男女均如此(补充图 S9C-D),但这并没有反映在血液或肿瘤中。在 B16-F10 模型中,NK 和 NKT 细胞非常重要:在男性中,CT 使血液中的 NK 细胞减少,肿瘤中的 NK 细胞增加(补充图 S9E),而 II 期 NKT 细胞仅在肿瘤中增加(图 1F)。比较两种肿瘤模型,多柔比星减少了血液和淋巴结中的免疫细胞总数,而奥沙利铂没有减少(补充图 S9A 和 S17A)。这些结果表明,不同肿瘤类型的免疫系统有不同的整体反应,血液或淋巴结中的变化并不反映肿瘤中观察到的变化,这与之前的报道[10]不同。我们首次在这一过程中观察到了具有相关临床意义的性别二态性。最后,我们发现不同的肿瘤模型表现出不同的免疫反应,因此禁食对化疗的促进作用可能并不取决于特定的免疫群体(图1O)。曼努埃尔-蒙特罗-戈麦斯-德拉斯-赫拉斯(Manuel Montero Gómez de las Heras)和何塞-伊格纳西奥-埃斯克里格-拉雷纳(Jose Ignacio Escrig-Larena):高维免疫方法学;形式分析和调查。Marta Barradas:免疫荧光研究设计与分析。克里斯蒂娜-潘托哈(Cristina Pantoja):项目管理、数据采集和整理以及小鼠样本处理。Adrian Plaza:小鼠样本处理、调查和数据解读。Jose Luis Lopez-Aceituno:小鼠样本处理和技术优化。Esther Durán:免疫荧光研究的样本处理。Alejo Efeyan:为免疫荧光实验提供支持。Maria Mittelbrunn:支持高维细胞测量实验。Andrés Pastor Fernández曾获得西班牙抗癌协会(AECC)的博士前奖学金(PRDMA18011PAST)。克里斯蒂娜-潘托哈(Cristina Pantoja)和玛尔塔-巴拉达斯(Marta Barradas)获得了马德里高等研究所(IMDEA)食品部的资助。Adrián Plaza 获得了马德里高等研究所(AECC)(SIRTBIO-LABAE18008FERN)的资助。Jose Luis Lopez-Aceituno 由西班牙科学与创新部(MICINN)(PTA2017-14689-I)资助。巴勃罗-何塞-费尔南德斯-马科斯(Pablo Jose Fernandez-Marcos)获得了西班牙科学、创新和大学部(MICINN)的拉蒙-卡哈尔奖(RYC-2017-22335 /AEI/10.13039/501100011033)的资助。巴勃罗-何塞-费尔南德斯-马科斯实验室的工作得到了 AECC(SIRTBIO- LABAE18008FERN)和 MICINN 的 RETOS 计划项目(SAF2017-85766-R/AEI/10.
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来源期刊
Cancer Communications
Cancer Communications Biochemistry, Genetics and Molecular Biology-Cancer Research
CiteScore
25.50
自引率
4.30%
发文量
153
审稿时长
4 weeks
期刊介绍: Cancer Communications is an open access, peer-reviewed online journal that encompasses basic, clinical, and translational cancer research. The journal welcomes submissions concerning clinical trials, epidemiology, molecular and cellular biology, and genetics.
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