ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

AS acknowledges the Immunology Training Grant, T32 AI007363. KSK gratefully acknowledges the Schlumberger Foundation for supporting her through the Faculty for the Future program.

All procedures were conducted with the approval of, and in compliance with, the Institutional Animal Care and Use Committee (IACUC) guidelines5. All animal procedures were performed in Dartmouth&#8217;s Animal Facility. Female C57BL/6 mice, 6&#8211;8 weeks of age, were housed under standard conditions on ventilated racks with food and water. The reagents and the equipment used are listed in the Table of Materials.&#160;
1. Shaving mice 
NOTE: All personnel must complete institutional training in live animal handling and anesthesia prior to performing these procedures.
<ol>
	<li>Prior to intradermal injection, shave the flank of each mouse as described below; the right flank was used for injections and the described procedures. Shave the mice the day prior to injecting tumor cells.</li>
	<li>Position the mouse on a stable surface. Scruff the mouse by the skin of the nape with the left thumb and index finger, and gently tuck the left side of the body into the palm to expose the right side. Use the ring and/or pinky fingers to hold the left leg and tail, ensuring the right flank is fully exposed.</li>
	<li>Shave the fur from the hind leg up to the lower ribs to create a clear area for injection.</li>
	<li>Both flanks may be shaved if additional practice space is needed. 
	NOTE: Beginners can anesthetize mice when learning to shave and properly scruff.</li>
</ol>
2. Practice injections with dye
NOTE: Practice can be performed on both live&#160;(anesthetized) and euthanized mice, and on either sex. Male mice typically have thicker and less pliable skin compared to females, making the procedure more technically challenging. For this reason, practicing on males is useful for developing proficiency.
<ol>
	<li>Mix food coloring with phosphate-buffered saline (PBS) in a 1.5 mL Eppendorf tube until the solution appears dark (blue food coloring was used in this study).</li>
	<li>Obtain practice mice and anesthetize them in accordance with institutional animal care guidelines. Apply ophthalmic ointment to both eyes to prevent corneal drying.</li>
	<li>Place the anesthetized mouse under a nose cone and tilt the body to expose the right flank. Confirm adequate anesthesia by gently pinching the footpad and observing no response.</li>
	<li>Load 30 &#181;L of dye solution into a 0.3 mL insulin syringe with a 29 G, &#189;&#8221; needle. Remove all air bubbles.</li>
	<li>Using the non-dominant hand, gently pinch and stretch the leg/skin to create a smooth, taut surface (avoid lifting the skin upward). Insert the needle bevel-up into the dermis at a shallow parallel angle to the skin. Keep the needle visible beneath the surface to confirm dermal placement. 
	NOTE: During practice, test different hand grip positions to maintain stable skin tension while keeping fingers clear of the needle path. Insert the needle ~2 mm into the skin. To reduce backflow, insert the needle ~3 mm, then withdraw slightly to create a &#8220;pocket&#8221; before injection. This helps retain the dye at the injection site.</li>
	<li>Slowly inject all 30 &#181;L of the dye solution until a small, distinct &#8220;bubble&#8221; forms under the skin surface 
	NOTE: The absence of visible bubbles suggests that the injection was delivered subcutaneously rather than intradermally. Maintaining the bevel in an upward position helps ensure accurate dermal placement and prevents overly deep injection.</li>
	<li>Continue practicing until consistently forming intradermal bubbles. Up to six to eight injections can be performed per side.</li>
	<li>After completing practice injections, euthanize the mice in accordance with approved Institutional Animal Care and Use Committee (IACUC) protocols5.</li>
</ol>
3. Preparation of tumor cells for injection
NOTE: Perform all cell-related steps in a sterile hood. Be mindful that different murine tumor lines grow differently.
<ol>
	<li>Thaw tumor cells (B16F10 used in this study) several days before intradermal injections.</li>
	<li>Culture cells to 70%&#8211;80% confluence while maintaining adequate fresh media 
	NOTE: Avoid 100% confluence, which halts proliferation and impairs tumor growth.</li>
	<li>Harvest and prepare cells at 7 x 106 cells/mL in either 1x PBS, serum-free RPMI, or other media used for growing the cells. 
	NOTE: The B16F10 tumor model used in the lab uses 2 x 105 cells in 30 &#181;L. The 30 &#181;L injection volume is appropriate, but the number of cells can be adjusted as needed to establish the desired tumor model. Cells will settle and collect at the bottom of the tube, so to ensure the concentration of cells is constant, periodically gently mix the cells to keep them suspended.</li>
	<li>Keep cells on ice after processing&#160;and proceed quickly to inject. Prolonged storage on ice reduces cell viability, which can decrease the number of live cells delivered and negatively impact tumor establishment6. 
	NOTE: Cells are harvested and used right away in the lab. Injections are done within 20&#8211;60 min of processing the cells and putting them on ice. Use a partner when working with large cohorts to minimize time on ice.</li>
</ol>
4. Intradermal injections 
<ol>
	<li>Anesthetize experimental mice.&#160;Apply ophthalmic ointment to both eyes to prevent corneal drying. 
	NOTE: Anesthesia with isoflurane gas is used by the lab, but injectable anesthetics can be used.</li>
	<li>Following anesthesia induction, place the mouse under a nose cone and position the body to fully expose the right flank.</li>
	<li>Gently invert the cell suspension to ensure even mixing.</li>
	<li>Draw 30 &#181;L of cell suspension into a 0.3 mL syringe with a 29 G, &#189;&#8221; needle. Remove air bubbles.</li>
	<li>Using the non-dominant hand, gently stretch the leg/skin to the side to make it smooth and tight (do not lift it upward). Insert the needle bevel-up into the dermis at a shallow parallel angle. The needle should stay visible under the skin, showing it&#8217;s not too deep. 
	NOTE: The needle depth should be ~2 mm into the skin. To avoid backflow of the cells, insert the needle ~3 mm in the skin, pull the needle back slightly to create a &#8220;pocket&#8221; for the cells to be injected into. This reduces backflow significantly.</li>
	<li>Very slowly inject all 30 &#181;L of cell suspension until a small, visible &#8220;bubble&#8221; forms under the skin. Wait 5&#8211;10 s before removing the needle. 
	NOTE: Removing the needle too quickly can cause the solution to come out of the skin due to the pressure.</li>
	<li>Repeat for each mouse. Replace syringes after each cage (4&#8211;5 mice) to prevent needle dulling.</li>
	<li>Monitor all mice until they have fully recovered from anesthesia.</li>
	<li>Monitor the mice for tumor establishment and overall health.</li>
</ol>
5. Intratumoral injections 
NOTE: Tumors are typically ready for treatment 7&#8211;10 days post-implantation, once they reach the appropriate size. Appropriate size used in the lab is ~50 mm3 or higher using the formula: Volume = (Length/2) * (Width*Width)7,8.
<ol>
	<li>Prepare all treatment solutions in advance (e.g., 1x PBS was used in this study). Keep solutions on ice or at room temperature as required by the specific treatment.</li>
	<li>Induce anesthesia according to institutional guidelines.</li>
	<li>Place the mouse under a nose cone to maintain anesthesia, and position it so the tumor is exposed.</li>
	<li>Draw the required volume (e.g., 30 &#181;L used here) into a 0.3 mL insulin syringe with a 29 G, &#189;&#8221; needle. Remove air bubbles. 
	NOTE: Volume will differ based on the treatment of choice and optimized conditions.</li>
	<li>Insert the needle horizontally into the tumor and advance toward the center of the tumor mass. 
	NOTE: Avoid inserting from the top or at a steep angle, as this can result in the injection being delivered too deep or into surrounding tissue. The treatment described was administered as a single injection. The doses and frequency are all dependent on the treatment of choice and optimized conditions. With small tumors, a single dose is adequate in distributing the treatment, but with larger tumors, multiple injections may be needed to distribute the treatment equally through the tumor mass.</li>
	<li>Inject the treatment slowly and steadily. Pause for 10 s before slowly withdrawing the needle to minimize backflow or leakage. Some tumors have high internal pressure, and care should be taken to ensure the injection does not leak out through the needle hole.</li>
	<li>Repeat the procedure for each mouse. Replace syringes after each cage of 4&#8211;5 mice and when switching treatment groups to prevent cross-contamination.</li>
	<li>Monitor mice post-anesthesia until they regain mobility and normal respiration. Continue daily monitoring for treatment response and overall health. 
	NOTE: Be mindful that different cell lines may cause tumors to ulcerate at different times. Each experiment and each IACUC protocol has clearly stated endpoints and exclusion criteria. In the lab, endpoints for mouse experiments are 1500 mm3 and ulcerations that occur are accepted and not excluded from the study.</li>
</ol>

<ol>
	<li>Zhang, W., Moore, L., Ji, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+cancer+research.">Mouse models for cancer research.</a> Chin J Cancer. 30 (3), 149-152 (2011).</li><li>Silihe Kamga, K., Fiering, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+immunotherapy+prior+to+cancer+surgery+a+promising+therapeutic+approach.">Intratumoral immunotherapy prior to cancer surgery a promising therapeutic approach.</a> Front Immunol. 16, 1545000 (2025).</li><li>Carlson, P. M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depth+of+tumor+implantation+affects+response+to+in+situ+vaccination+in+a+syngeneic+murine+melanoma+model.">Depth of tumor implantation affects response to in situ vaccination in a syngeneic murine melanoma model.</a> J Immunother Cancer. 9 (4), e002107 (2021).</li><li>Bonnotte, B., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+injection+as+opposed+to+subcutaneous+injection+enhances+immunogenicity+and+suppresses+tumorigenicity+of+tumor+cells.">Intradermal injection as opposed to subcutaneous injection enhances immunogenicity and suppresses tumorigenicity of tumor cells.</a> Cancer Res. 63 (9), 2145-2149 (2003).</li><li>Garber, J. C., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Ya, Z., Hailemichael, Y., Overwijk, W., Restifo, N. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+for+pre-clinical+study+of+human+cancer+immunotherapy.">Mouse model for pre-clinical study of human cancer immunotherapy.</a> Curr Protoc Immunol. 109, 20.1.1-20.1.43 (2015).</li><li>Mao, C., Beiss, V., Ho, G. W., Fields, J., Steinmetz, N. F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ.+vaccination+with+cowpea+mosaic+virus+elicits+systemic+antitumor+immunity+and+potentiates+immune+checkpoint+blockade.">In situ. vaccination with cowpea mosaic virus elicits systemic antitumor immunity and potentiates immune checkpoint blockade.</a> J Immunother Cancer. 10 (12), e005834 (2022).</li><li>Ho, G. W., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+expression+of+IL-12+and+CD40+ligand+(CD154)+from+plasmids+generates+antitumor+responses+that+eliminate+tumoral+T+regs.">Intratumoral expression of IL-12 and CD40 ligand (CD154) from plasmids generates antitumor responses that eliminate tumoral T regs.</a> Sci Rep. 15 (1), 24439 (2025).</li><li>Kim, Y. C., Jarrahian, C., Zehrung, D., Mitragotri, S., Prausnitz, M. R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+systems+for+intradermal+vaccination.">Delivery systems for intradermal vaccination.</a> Curr Top Microbiol Immunol. 351, 77-112 (2012).</li><li>Chen, X. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+adjuvants+for+intradermal+vaccination.">Emerging adjuvants for intradermal vaccination.</a> Int J Pharm. 632, 122559 (2023).</li><li>Hickling, J. K., Jones, K. R., Friede, M., Zehrung, D., Chen, D., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intradermal+delivery+of+vaccines:+potential+benefits+and+current+challenges.">Intradermal delivery of vaccines: potential benefits and current challenges.</a> Bull World Health Organ. 89 (3), 221-226 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

The success of this protocol (Figure 4) depends on maintaining a consistent intradermal injection depth and precise needle placement to ensure delivery into the dermis rather than the subcutaneous layer. If the needle angle is too steep or the injection pressure is too high, the dye or tumor cells may be deposited through the skin into the subcutaneous space. After injection, pausing briefly before slowly withdrawing the needle helps prevent leakage or formation of doublet tumors (&#8220;sno...

Murine tumor models constitute an indispensable tool in cancer research. In an optimal mouse model, it would be possible to induce tumors that closely mimic human tumors at both genetic and morphologic levels1. This enables the assessment and efficiency of novel therapeutic approaches in different fields, including intratumoral immunotherapy (ITIT). ITIT is an emerging field in cancer treatment that consists of directly administering immune-stimulating agents into the tumor micro-environment (TME). This localized delivery can enhance local immune activation, reverse the immunosuppressive tumor micro-environment, and trigger systemic antitumor immune responses, known as the abscopal effect2. However, the efficiency, accuracy, and consistency of intratumoral administration depend on the ability to visualize and control injection parameters in different tumor models.
Most pre-clinical cancer studies use subcutaneous tumor implantation. However, this approach has limitations for intratumoral therapies because the tumor cannot be directly visualized during injection unless surgery is performed. Many intratumoral immunotherapy strategies require precise delivery of reagents into small mouse tumors and often entail accomplishing that task multiple times. Without visual confirmation, injection accuracy and reagent retention within subcutaneous tumors become poorly controlled variables. This could result in misinterpretation/variation of experimental data3.
Intradermal tumor implantation provides a direct, accessible, visualizable, and reproducible alternative for tumor observation, measurement, and intratumoral injection, thus enhancing consistency. Moreover, studies have shown that tumors implanted intradermally, as opposed to subcutaneous injections, enhance immunogenicity and suppress the tumorigenicity of tumor cells4. Intradermally injected tumors induced rapid and strong dendritic cell trafficking to lymph nodes, resulting in earlier and more robust activation of tumor-specific cytotoxic T cells4. The present study describes a detailed and reproducible protocol for establishing intradermal B16F10 melanoma tumors in C57BL/6 mice and performing subsequent intratumoral treatments.

<table><tbody><tr><td>1.5 mL Natural Centrifuge Tube</td><td>USA Scientific&amp;nbsp;</td><td>1615-5500</td><td></td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; Cell Culture Flask</td><td>Corning</td><td>430825</td><td></td></tr><tr><td>Fluriso</td><td>Vet One</td><td>13985-528-60</td><td>isoflurane</td></tr><tr><td>Food Coloring</td><td>Durkee</td><td></td><td>for practice</td></tr><tr><td>Insulin syringe 0.33 x 12.7 mm</td><td>Exel</td><td>26018</td><td></td></tr><tr><td>Mini Arco</td><td>Wahl</td><td>08787-450A</td><td>clippers to shave the mice</td></tr><tr><td>PBS, 1X w/o calcium &amp;amp; magnesium</td><td>Corning</td><td>06325002</td><td>pH 7.4 +/- 0.1</td></tr><tr><td>RPMI 1640, 1x</td><td>Corning</td><td>12225004</td><td></td></tr><tr><td>SomnoFlo Low-Flow Electronic Vaporizer</td><td>Kent Scientific&amp;nbsp;</td><td>SF-01</td><td>anesthesia machine</td></tr></tbody></table>

intradermal tumor implantation as a robust model for murine cancer intratumoral immunotherapy studies

Intradermal Tumor Implantation as a Robust Model for Murine Cancer Intratumoral Immunotherapy Studies

Establishing reproducible murine tumor models is essential for evaluating cancer biology and immunotherapy strategies. Intratumoral immunotherapy treats established tumors with immune-stimulating reagents to reverse local immune suppression and stimulate systemic antitumor immunity. Most cancer studies in mice establish the tumors in the subcutaneous space, which has inherent challenges for experimental intratumoral immunotherapy due to the inability to visualize intratumoral injections. Intradermal injection of tumor cells provides a reliable approach to generate visualizable tumors that support accurate and reproducible intratumoral injection and monitoring of growth kinetics and therapeutic responses. Here, we present a reproducible methodology for establishing and treating intradermal tumors using the murine B16F10 melanoma model in C57BL/6 mice as our example. Tumor cell suspensions are injected intradermally, and 7&#8211;10 days after tumor implantation, once tumors reach ~60 mm3, an intratumoral injection of immune-stimulating agents or a physical treatment like electroporation or heating can be directly applied. This allows precise delivery of treatments and measurement of tumor growth with calipers and avoids the challenge of the inherent variability of multiple treatments of subcutaneous tumors that cannot be visualized. By providing detailed insight into this technique, this article aims to support reproducibility and advance pre-clinical research in intratumoral immunotherapy.

When practicing injections with dye, successful intradermal delivery can be verified without euthanizing the mouse by observing the formation of a visible “bubble” at the injection site. A properly performed intradermal injection produces a large, well-defined, and raised “bubble” (Figure 1A–F). Small or flat bubbles suggest that the injection may be partially subcutaneous. If no “bubble” forms and the dye or cells spread beneath the skin, the injection was likely subcutaneo...

With increasing interest in intratumoral immunotherapy, selecting tumor models that are accessible and practical is essential. Subcutaneous tumors are easy to establish but difficult to visualize and manipulate. In this protocol, we describe methods for generating intradermal tumors that provide improved visibility and accessibility.

Establishing reproducible murine tumor models is essential for evaluating cancer biology and immunotherapy strategies. Intratumoral immunotherapy treats ...

intradermal-tumor-implantation-as-robust-model-for-murine-cancer

Research

JoVE Journal

Cancer Research

In this video, human-derived melanoma cells are implanted subcutaneously into the flank region of an immunocompromised mouse. The PDX model allows preclinical investigation of melanoma cells that better recapitulate the tumor heterogeneity and melanoma aggressiveness observed in in vivo conditions.

Patient-derived Xenograft Modeling: A Technique to Generate Melanoma Mouse Models

In this video, we develop an orthotopic head and neck cancer murine model by intramucosal inoculation of the squamous cell carcinoma cell line. The tumor-bearing mouse would show resemblance to clinically relevant symptoms of human disease and can thus be used to study cancer progression and discover novel therapeutic strategies.

Intramucosal Inoculation of Cancer Cells in Murine Model: A Procedure to Establish an Orthotopic Head and Neck Cancer Mouse Model

This video demonstrates a cell-based tumor vaccination assay. A mouse, intradermally injected with B16-F10 melanoma cells for tumor formation, is vaccinated with B16-F10 cells secreting Flt3L cytokine near the developed tumor. The secreted Flt3L from the vaccine promotes the maturation and proliferation of dendritic cells that then present tumor antigens to T cells in lymph nodes, triggering a targeted anti-tumor immune response.

Tumor Implantation and Injection of a Tumor Vaccine in a Mouse Model

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Dartmouth Geisel School of Medicine.  With increasing interest in intratumoral immunotherapy, selecting tumor models that are accessible and practical is essential. Subcutaneous tumors are easy to establish but difficult to visualize and manipulate. In this protocol, we describe methods for generating intradermal tumors that provide improved visibility and accessibility.

Intradermal Tumor Implantation as a Robust Model for Murine Cancer Intratumoral Immunotherapy Studies

Explore More Videos