Cancer stem cells and metabolic reprogramming in breast cancer

La síntesis de novo de colesterol y su enzima limitante, hidroximetilglutaril-coenzima A reductasa (HMGCR), es crítica para la regulación de la proliferación y la supervivencia celular en cáncer. El objetivo general del presente trabajo es estudiar el papel de la vía del colesterol en la adquisición de propiedades de células madre en modelos de cáncer de mama (CM). Con ese propósito, se analizaron bases de datos públicas, compiladas de pacientes con CM, mediante una herramienta bioinformática online (SurvExpress; Tecnológico de Monterrey, México). Los resultados muestran que la alta expresión de varias enzimas involucradas en la síntesis del colesterol, incluyendo a: hidroximetilglutaril-CoA sintasa 1 (HMGCS1), HMGCR, mevalonato quinasa (MVK), farnesil pirofostato sintasa (FDPS) y escualeno monooxigenasa (SQLE), está significativamente asociada con menor sobrevida libre de enfermedad. Adicionalmente, la herramienta bioinformática online CellMiner (Instituto Nacional del Cáncer de los Estados Unidos) que integra datos de expresión génica y farmacológicos provenientes del panel de líneas celulares NCI-60, indica que HMGCR se expresa en varios tipos de tumores, incluyendo CM. Estos datos se validaron experimentalmente por qRT-PCR. Para evaluar si existe una asociación entre la expresión de HMGCR y características de células madre, se desarrolló un modelo de sobreexpresión de HMGCR en la línea celular MCF-7 utilizando un sistema CRISPR activador (CRISPRon). La generación de dicho sistema comprendió las siguientes etapas: 1-diseño y síntesis de las sondas (sgRNAs); 2-clonación de las sondas; 3-transformación de bacterias con los plásmidos clonados; 4-verificación de dichas construcciones por secuenciación; 5- transfección de líneas celulares; 6-caracterización del sistema. La expresión de HMGCR aumenta significativamente en las células MCF-7/CR en comparación al control de transfección (MCF-7/TC) y se observan niveles similares a los detectados en otros modelos de células madre (normales y tumorales). El fenotipo HMGCRon se asocia a un incremento de los niveles de los marcadores de pluripotencia Nanog y Sox2, un aumento en la población CD44+/CD24- y CD133+ [fórmula aproximada, revisar la misma en el original] y un aumento en la frecuencia de formación de mamoesferas, características asociadas a células madre tumorales (CMTs) en CM. A continuación, se evaluó la respuesta de líneas celulares de CM con distintos niveles de HMGCR al tratamiento con las estatinas simvastatina (SIM) y lovastatina (LOVA). Los resultados muestran una mayor sensibilidad en las líneas basales Hs578T y MDA-MB-231 (CM humano triple negativo) en comparación con las líneas luminales MCF-7 y T47D [CM humano, receptor de estrógeno (ER) y progesterona (PR) positivo, receptor del factor de crecimiento epidérmico HER2/neu negativo], que son más resistentes. Estos datos fueron corroborados con la herramienta CellMiner. Sin embargo, la línea T47D que expresa altos niveles de HMGCR es más sensible a SIM que MCF-7, indicando que en un contexto ER positivo, la sensibilidad a estatinas podría estar parcialmente determinada por HMGCR. Esto se corroboró en el sistema HMGCRon, ya que las células MCF-7/CR muestran mayor sensibilidad a SIM que la línea control MCF-7/TC. Estos datos sugieren que la expresión de HMGCR está asociada a características de CMTs y determina, al menos en parte, la sensibilidad a estatinas, sentando las bases para estudios posteriores orientados a una terapéutica anti-colesterol específica para el compartimiento de células madre en CM.The de novo synthesis of cholesterol, and its rate-limiting enzyme, hydroxymethylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stemness in both transformed and non-transformed cells is still unclear. First, we analyzed public compiled databases from breast cancer (BC) patients with the web application SurvExpress from the Tecnológico de Monterrey, México. We found that high expression levels of several members of the mevalonate (MVA) pathway, including hydroxymethylglutharyl-coenzyme A synthase 1 (HMGCS1), HMGCR, mevalonate kinase (MVK), Farnesyl pyrophosphate synthase (FDPS) and squalene monooxygenase (SQLE), were associated with a lower recurrence-free survival. The web application Cell Miner from the National Cancer Institute from the United States (NCI), that allows the integration of genomic and pharmacological data, showed that HMGCR is expressed in several cancer cell lines, including BC. These data were further validated by qRT-PCR in a breast cancer cell line panel. With the purpose of endogenously increasing HMGCR expression levels, we took advantage of the system CRISPR/on, which includes small guide RNA (sgRNA) expression vectors (pSPgRNA) and the plasmid carrying the sequence for dCas9-VP160. Briefly, these steps were followed for the development of an HMGCRon system: 1-sgRNAs design and synthesis; 2-sgRNAs cloning; 3-bacteria transformation with the constructs carrying the sgRNAs; 4-validation of the constructs by sequencing; 5-transfection of the cell line MCF-7; 6-characterization of the system. The CRISPRon system significantly increased HMGCR total levels in MCF-7 cells (MCF-7/CR) when compared to transfection controls (MCF-7/TC), and were similar to those observed in other stem cell lines, both transformed and non-transformed. Additionally, the induction of an HMGCRon phenotype increased the pluripotency markers Nanog and Sox2, cancer stem cell traits such as the CD44+/CD24 low/- and the CD133+ [fórmula aproximada, revisar la misma en el original] populations and the formation of mammospheres. Next, we assessed the sensitivity of BC-derived cell lines expressing different levels of HMGCR to the statins simvastatin (SIM) and lovastatin (LOVA). Hs578T cells and MDA-MB-231 (triple negative human BC cell lines) were sensitive to both drugs, whereas MCF-7 and T47D [Estrogen Receptor (ER) and Progesterone Receptor (PR) negative, human epidermal growth factor 2 (HER2/neu) negative] cells were more resistant. These data was also confirmed with the CellMiner tool. Interestingly, T47D cells, which express high levels of HMGCR, were more sensitive to SIM than MCF-7 cells, suggesting that in ER-positive cells HMCGR would be, at least in part, determining the sensitivity towards statins. This was further corroborated since the induction of an HMGCRon phenotype in MCF-7 cells increased the sensitivity to SIM. Altogether, these data suggest that expression of HMGCR is associated with stem cell traits in BC and determines sensitivity to statin in some cases, encouraging further studies of the use of anti-cholesterol drugs as therapies to target the stem compartment of the tumor.Fil: Marks, María Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Cancer stem cells and metabolic reprogramming in breast cancer

La síntesis de novo de colesterol y su enzima limitante, hidroximetilglutaril-coenzima A reductasa (HMGCR), es crítica para la regulación de la proliferación y la supervivencia celular en cáncer. El objetivo general del presente trabajo es estudiar el papel de la vía del colesterol en la adquisición de propiedades de células madre en modelos de cáncer de mama (CM). Con ese propósito, se analizaron bases de datos públicas, compiladas de pacientes con CM, mediante una herramienta bioinformática online (SurvExpress; Tecnológico de Monterrey, México). Los resultados muestran que la alta expresión de varias enzimas involucradas en la síntesis del colesterol, incluyendo a: hidroximetilglutaril-CoA sintasa 1 (HMGCS1), HMGCR, mevalonato quinasa (MVK), farnesil pirofostato sintasa (FDPS) y escualeno monooxigenasa (SQLE), está significativamente asociada con menor sobrevida libre de enfermedad. Adicionalmente, la herramienta bioinformática online CellMiner (Instituto Nacional del Cáncer de los Estados Unidos) que integra datos de expresión génica y farmacológicos provenientes del panel de líneas celulares NCI-60, indica que HMGCR se expresa en varios tipos de tumores, incluyendo CM. Estos datos se validaron experimentalmente por qRT-PCR. Para evaluar si existe una asociación entre la expresión de HMGCR y características de células madre, se desarrolló un modelo de sobreexpresión de HMGCR en la línea celular MCF-7 utilizando un sistema CRISPR activador (CRISPRon). La generación de dicho sistema comprendió las siguientes etapas: 1-diseño y síntesis de las sondas (sgRNAs); 2-clonación de las sondas; 3-transformación de bacterias con los plásmidos clonados; 4-verificación de dichas construcciones por secuenciación; 5- transfección de líneas celulares; 6-caracterización del sistema. La expresión de HMGCR aumenta significativamente en las células MCF-7/CR en comparación al control de transfección (MCF-7/TC) y se observan niveles similares a los detectados en otros modelos de células madre (normales y tumorales). El fenotipo HMGCRon se asocia a un incremento de los niveles de los marcadores de pluripotencia Nanog y Sox2, un aumento en la población CD44+/CD24- y CD133+ [fórmula aproximada, revisar la misma en el original] y un aumento en la frecuencia de formación de mamoesferas, características asociadas a células madre tumorales (CMTs) en CM. A continuación, se evaluó la respuesta de líneas celulares de CM con distintos niveles de HMGCR al tratamiento con las estatinas simvastatina (SIM) y lovastatina (LOVA). Los resultados muestran una mayor sensibilidad en las líneas basales Hs578T y MDA-MB-231 (CM humano triple negativo) en comparación con las líneas luminales MCF-7 y T47D [CM humano, receptor de estrógeno (ER) y progesterona (PR) positivo, receptor del factor de crecimiento epidérmico HER2/neu negativo], que son más resistentes. Estos datos fueron corroborados con la herramienta CellMiner. Sin embargo, la línea T47D que expresa altos niveles de HMGCR es más sensible a SIM que MCF-7, indicando que en un contexto ER positivo, la sensibilidad a estatinas podría estar parcialmente determinada por HMGCR. Esto se corroboró en el sistema HMGCRon, ya que las células MCF-7/CR muestran mayor sensibilidad a SIM que la línea control MCF-7/TC. Estos datos sugieren que la expresión de HMGCR está asociada a características de CMTs y determina, al menos en parte, la sensibilidad a estatinas, sentando las bases para estudios posteriores orientados a una terapéutica anti-colesterol específica para el compartimiento de células madre en CM.The de novo synthesis of cholesterol, and its rate-limiting enzyme, hydroxymethylglutharyl-coenzyme A reductase (HMGCR), is deregulated in tumors and critical for tumor cell survival and proliferation. However, the role of HMGCR in the induction and maintenance of stemness in both transformed and non-transformed cells is still unclear. First, we analyzed public compiled databases from breast cancer (BC) patients with the web application SurvExpress from the Tecnológico de Monterrey, México. We found that high expression levels of several members of the mevalonate (MVA) pathway, including hydroxymethylglutharyl-coenzyme A synthase 1 (HMGCS1), HMGCR, mevalonate kinase (MVK), Farnesyl pyrophosphate synthase (FDPS) and squalene monooxygenase (SQLE), were associated with a lower recurrence-free survival. The web application Cell Miner from the National Cancer Institute from the United States (NCI), that allows the integration of genomic and pharmacological data, showed that HMGCR is expressed in several cancer cell lines, including BC. These data were further validated by qRT-PCR in a breast cancer cell line panel. With the purpose of endogenously increasing HMGCR expression levels, we took advantage of the system CRISPR/on, which includes small guide RNA (sgRNA) expression vectors (pSPgRNA) and the plasmid carrying the sequence for dCas9-VP160. Briefly, these steps were followed for the development of an HMGCRon system: 1-sgRNAs design and synthesis; 2-sgRNAs cloning; 3-bacteria transformation with the constructs carrying the sgRNAs; 4-validation of the constructs by sequencing; 5-transfection of the cell line MCF-7; 6-characterization of the system. The CRISPRon system significantly increased HMGCR total levels in MCF-7 cells (MCF-7/CR) when compared to transfection controls (MCF-7/TC), and were similar to those observed in other stem cell lines, both transformed and non-transformed. Additionally, the induction of an HMGCRon phenotype increased the pluripotency markers Nanog and Sox2, cancer stem cell traits such as the CD44+/CD24 low/- and the CD133+ [fórmula aproximada, revisar la misma en el original] populations and the formation of mammospheres. Next, we assessed the sensitivity of BC-derived cell lines expressing different levels of HMGCR to the statins simvastatin (SIM) and lovastatin (LOVA). Hs578T cells and MDA-MB-231 (triple negative human BC cell lines) were sensitive to both drugs, whereas MCF-7 and T47D [Estrogen Receptor (ER) and Progesterone Receptor (PR) negative, human epidermal growth factor 2 (HER2/neu) negative] cells were more resistant. These data was also confirmed with the CellMiner tool. Interestingly, T47D cells, which express high levels of HMGCR, were more sensitive to SIM than MCF-7 cells, suggesting that in ER-positive cells HMCGR would be, at least in part, determining the sensitivity towards statins. This was further corroborated since the induction of an HMGCRon phenotype in MCF-7 cells increased the sensitivity to SIM. Altogether, these data suggest that expression of HMGCR is associated with stem cell traits in BC and determines sensitivity to statin in some cases, encouraging further studies of the use of anti-cholesterol drugs as therapies to target the stem compartment of the tumor.Fil: Marks, María Paula. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Assessing the distribution of cancer stem cells in tumorspheres

Abstract Cancer Stem Cells presumably drive tumor growth and resistance to conventional cancer treatments. From a previous computational model, we inferred that these cells are not uniformly distributed in the bulk of a tumorsphere. To confirm this result, we cultivated tumorspheres enriched in stem cells, and performed immunofluorescent detection of the stemness marker SOX2 using confocal microscopy. In this article, we present an image processing method that reconstructs the amount and location of the Cancer Stem Cells in the spheroids. Its advantage is the use of a statistical criterion to classify the cells in Stem and Differentiated, instead of setting an arbitrary threshold. Moreover, the analysis of the experimental images presented in this work agrees with the results from our computational models, thus enforcing the notion that the distribution of Cancer Stem Cells in a tumorsphere is non-homogeneous. Additionally, the method presented here provides a useful tool for analyzing any image in which different kinds of cells are stained with different markers

Post-surgery statin use contributes to favorable outcomes in patients with early breast cancer

Statins are a group of lipid-lowering drugs with pleiotropic effects that include, but are not limited to the inhibition of cholesterol synthesis resulting in a wide range of anti-inflammatory, anti-tumor, immunomodulatory, and anti-thrombotic properties. This study aimed to determine the impact of the prior to- or after breast surgery usage of statins on the tumor prognosis in breast cancer (BC) patients.Fil: Giorello, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Marks, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Osinalde, Tiago Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Padin, María del Rosario. Hospital Italiano; ArgentinaFil: Wernicke, Alejandra. Hospital Italiano; ArgentinaFil: Calvo, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Chasseing, Norma Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Vellón, Luciano. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

ROR1 contributes to melanoma cell growth and migration by regulating N-cadherin expression via the PI3K/Akt pathway

The Receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) is primarily expressed by neural crest cells during embryogenesis. Following a complete downregulation after birth, ROR1 was shown to re-express in various types of cancers. Little is known about ROR1 expression and function in melanoma. Here we show that ROR1 is aberrantly expressed in both melanoma cell lines and tumors and that its expression associates with poor Post-Recurrence Survival of melanoma. Using gain- and loss-of-function approaches we found that ROR1 enhances both anchorage-dependent and -independent growth of melanoma cells. In addition, ROR1 decreases cell adhesion and increases cell motility and migration. Mechanistically, ROR1 was found to induce upregulation of Akt and the mesenquimal markers N-cadherin and vimentin. The regulation of N-cadherin by ROR1 relies on both Akt dependent and independent mechanisms. ROR1 does not affect Wnt canonical pathway but was found to be engaged in a positive feedback loop with Wnt5a. In summary, we show that ROR1 contributes to melanoma progression and is a candidate biomarker of poor prognosis. Although further studies are needed to confirm this possibility, the present work indicates that ROR1 is a good prospective target for melanoma cancer therapy.Fil: Fernández, Natalia Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Lorenzo, Daniela Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Picco, María Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Barbero, Gastón Alexis. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico. Departamento de Estudios Biomédicos y Biotecnológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dergan Dylon, Leonardo Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Marks, Maria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Garcia Rivello, Hernan Jorge. Hospital Italiano; ArgentinaFil: Gimenez, Liliana. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Labovsky, Vivian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Grumolato, Luca. University Of Rouen; FranciaFil: Lopez Bergami, Pablo Roberto. Universidad Maimónides. Área de Investigaciones Biomédicas y Biotecnológicas. Centro de Estudios Biomédicos, Biotecnológicos, Ambientales y de Diagnóstico. Departamento de Estudios Biomédicos y Biotecnológicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain: a prospective observational cohort study.

BACKGROUND: In May, 2022, several European countries reported autochthonous cases of monkeypox, which rapidly spread globally. Early reports suggest atypical presentations. We aimed to investigate clinical and virological characteristics of cases of human monkeypox in Spain. METHODS: This multicentre, prospective, observational cohort study was done in three sexual health clinics in Madrid and Barcelona, Spain. We enrolled all consecutive patients with laboratory-confirmed monkeypox from May 11 to June 29, 2022. Participants were offered lesion, anal, and oropharynx swabs for PCR testing. Participant data were collected by means of interviews conducted by dermatologists or specialists in sexually transmitted infections and were recorded using a standard case report form. Outcomes assessed in all participants with a confirmed diagnosis were demographics, smallpox vaccination, HIV status, exposure to someone with monkeypox, travel, mass gathering attendance, risk factors for sexually transmitted infections, sexual behaviour, signs and symptoms on first presentation, virological results at multiple body sites, co-infection with other sexually transmitted pathogens, and clinical outcomes 14 days after the initial presentation. Clinical outcomes were followed up until July 13, 2022. FINDINGS: 181 patients had a confirmed monkeypox diagnosis and were enrolled in the study. 166 (92%) identified as gay men, bisexual men, or other men who have sex with men (MSM) and 15 (8%) identified as heterosexual men or heterosexual women. Median age was 37·0 years (IQR 31·0-42·0). 32 (18%) patients reported previous smallpox vaccination, 72 (40%) were HIV-positive, eight (11%) had a CD4 cell count less than 500 cells per μL, and 31 (17%) were diagnosed with a concurrent sexually transmitted infection. Median incubation was 7·0 days (IQR 5·0-10·0). All participants presented with skin lesions; 141 (78%) participants had lesions in the anogenital region, and 78 (43%) in the oral and perioral region. 70 (39%) participants had complications requiring treatment: 45 (25%) had a proctitis, 19 (10%) had tonsillitis, 15 (8%) had penile oedema, six (3%) an abscess, and eight (4%) had an exanthem. Three (2%) patients required hospital admission. 178 (99%) of 180 swabs from skin lesions collected tested positive, as did 82 (70%) of 117 throat swabs. Viral load was higher in lesion swabs than in pharyngeal specimens (mean cycle threshold value 23 [SD 4] vs 32 [6], absolute difference 9 [95% CI 8-10]; p<0·0001). 108 (65%) of 166 MSM reported anal-receptive sex. MSM who engaged in anal-receptive sex presented with proctitis (41 [38%] of 108 vs four [7%] of 58, absolute difference 31% [95% CI 19-44]; p<0·0001) and systemic symptoms before the rash (67 [62%] vs 16 [28%], absolute difference 34% [28-62]; p<0·0001) more frequently than MSM who did not engage in anal-receptive sex. 18 (95%) of 19 participants with tonsillitis reported practising oral-receptive sex. The median time from onset of lesions to formation of a dry crust was 10 days (IQR 7-13). INTERPRETATION: In our cohort, monkeypox caused genital, perianal, and oral lesions and complications including proctitis and tonsillitis. Because of the variability of presentations, clinicians should have a low threshold for suspicion of monkeypox. Lesion swabs showed the highest viral loads, which, combined with the history of sexual exposure and the distribution of lesions, suggests close contact is probably the dominant transmission route in the current outbreak. FUNDING: None

Clinical presentation and virological assessment of confirmed human monkeypox virus cases in Spain : a prospective observational cohort study

In May, 2022, several European countries reported autochthonous cases of monkeypox, which rapidly spread globally. Early reports suggest atypical presentations. We aimed to investigate clinical and virological characteristics of cases of human monkeypox in Spain. This multicentre, prospective, observational cohort study was done in three sexual health clinics in Madrid and Barcelona, Spain. We enrolled all consecutive patients with laboratory-confirmed monkeypox from May 11 to June 29, 2022. Participants were offered lesion, anal, and oropharynx swabs for PCR testing. Participant data were collected by means of interviews conducted by dermatologists or specialists in sexually transmitted infections and were recorded using a standard case report form. Outcomes assessed in all participants with a confirmed diagnosis were demographics, smallpox vaccination, HIV status, exposure to someone with monkeypox, travel, mass gathering attendance, risk factors for sexually transmitted infections, sexual behaviour, signs and symptoms on first presentation, virological results at multiple body sites, co-infection with other sexually transmitted pathogens, and clinical outcomes 14 days after the initial presentation. Clinical outcomes were followed up until July 13, 2022. 181 patients had a confirmed monkeypox diagnosis and were enrolled in the study. 166 (92%) identified as gay men, bisexual men, or other men who have sex with men (MSM) and 15 (8%) identified as heterosexual men or heterosexual women. Median age was 37·0 years (IQR 31·0-42·0). 32 (18%) patients reported previous smallpox vaccination, 72 (40%) were HIV-positive, eight (11%) had a CD4 cell count less than 500 cells per μL, and 31 (17%) were diagnosed with a concurrent sexually transmitted infection. Median incubation was 7·0 days (IQR 5·0-10·0). All participants presented with skin lesions; 141 (78%) participants had lesions in the anogenital region, and 78 (43%) in the oral and perioral region. 70 (39%) participants had complications requiring treatment: 45 (25%) had a proctitis, 19 (10%) had tonsillitis, 15 (8%) had penile oedema, six (3%) an abscess, and eight (4%) had an exanthem. Three (2%) patients required hospital admission. 178 (99%) of 180 swabs from skin lesions collected tested positive, as did 82 (70%) of 117 throat swabs. Viral load was higher in lesion swabs than in pharyngeal specimens (mean cycle threshold value 23 [SD 4] vs 32 [6], absolute difference 9 [95% CI 8-10]; p<0·0001). 108 (65%) of 166 MSM reported anal-receptive sex. MSM who engaged in anal-receptive sex presented with proctitis (41 [38%] of 108 vs four [7%] of 58, absolute difference 31% [95% CI 19-44]; p<0·0001) and systemic symptoms before the rash (67 [62%] vs 16 [28%], absolute difference 34% [28-62]; p<0·0001) more frequently than MSM who did not engage in anal-receptive sex. 18 (95%) of 19 participants with tonsillitis reported practising oral-receptive sex. The median time from onset of lesions to formation of a dry crust was 10 days (IQR 7-13). In our cohort, monkeypox caused genital, perianal, and oral lesions and complications including proctitis and tonsillitis. Because of the variability of presentations, clinicians should have a low threshold for suspicion of monkeypox. Lesion swabs showed the highest viral loads, which, combined with the history of sexual exposure and the distribution of lesions, suggests close contact is probably the dominant transmission route in the current outbreak