The authors wish you to thank Kim Yates, Eileen Franklin, and Rebecca Tjepkema (Animal Care and Veterinary Services, University of Ottawa) for assistance with animal surgeries.&#160;Lee-Hwa Tai is supported by a Fonds de recherche sant&#233; Quebec Fellowship.&#160; This work was supported by operating grants from the Canadian Cancer Society Research Institute Innovation Grant, Ontario Ministry of Research and Development Early Researcher Award, and Canadian Foundation for Innovation &#8211; Leader&#8217;s Opportunity Grant to Rebecca Auer.

1. Maintaining 4T1 Tumor Cells in vitro
<ol>
	<li>Culture unmodified 4T1 tumor cells in complete DMEM (DMEM, 10% FBS, 1x Penicillin/Streptomycin) in 10 cm tissue culture plates. Incubate in 37 &#176;C, 5% CO2 tissue culture incubator.</li>
	<li>Split cultures 2-3 times/week. To maintain optimum viability cells should not exceed 80% confluence. Do not use cells for establishing in vivo primary tumors if cultures have been passaged in vitro for &#62;1 month as this decreases malignancy and metastatic potential than earlier passaged cells.</li>
</ol>
2. Harvesting 4T1 Tumor Cells for Injection
<ol>
	<li>Aspirate culture medium from the tissue culture plate using a Pasteur pipette.</li>
	<li>Add 10 ml of 1x&#160;sterile PBS containing 2 ml EDTA. Let the PBS solution incubate in the plate at 37 &#176;C, 5% CO2 tissue culture incubator for 5-7 min.</li>
	<li>Harvest PBS solution from plate, rinsing the plate 2-3x with solution, transfer to 15 ml conical tube.</li>
	<li>Centrifuge cells for 5 min&#160;at 500 x g, at 4 &#176;C, in a bench-top centrifuge.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in serum-free DMEM.</li>
	<li>Determine cell concentration using a cell counter.</li>
	<li>Dilute cells with serum-free medium to 1x106 cells/500 &#956;l (1x105 cells/50 &#956;l (for injection and place cells on ice.</li>
</ol>
3. Injecting Mice with 4T1 Tumor Cells
All animal studies performed were in accordance with institutional guidelines at the Animal Care Veterinary Services of the University of Ottawa&#160;recommendations for all animals receiving orthotopic injections or tumor implants.
<ol>
	<li>Treat mice subcutaneously with Buprenorphine (0.05 mg/kg) 1 hr before surgery for pain management.</li>
	<li>Induce and maintain mouse under anesthesia using 2.5% isoflurane during the duration of the injection. Pinch the footpad of the mouse to detect reflex response. If none is detected, effective anesthesia levels are reached.&#160;Sterile eye lubricant is then applied to prevent corneal drying.</li>
	<li>Load a 30 G&#160;&#189; in&#160;&#34;Ultra-fine&#34; Syringe with precisely 50 &#956;l of cells suspension. Ensure that all air bubbles are removed from the syringe column by tapping the side of the syringe to dislodge air bubbles.</li>
	<li>Place mouse (under anesthesia) ventral side up.</li>
	<li>Clean the injection site using an alcohol swab and introduce the needle horizontally and directly into the 4th mammary fat pad, slowly dispense the syringe volume.</li>
	<li>Use a cotton swab to clean any possible leakage.</li>
	<li>Allow mice to recover from anesthesia.</li>
	<li>Maintain Buprenorphine (0.05 mg/kg) for pain management administered s.c. every 8 hr for 2 days.</li>
</ol>
4. Administering Perioperative Treatment in 4T1 Tumor Bearing BALB/c Mice
&#160; &#160; &#160; &#160; &#160; &#160;The treatment regimen and route for influenza vaccine administration is identical to the oncolytic virus preoperative treatment.
<ol>
	<li>At 13 days post-tumor cell injection, measure the primary tumor using an external caliper. Measure the greatest longitudinal diameter (length) and the greatest transverse diameter (width) with the caliper. The modified ellipsoidal formula is used to calculate the tumor volume (Tumor volume = 1/2(length &#195;&#8212; width2)</li>
	<li>At 13 days post-tumor cell injection, the primary tumor should measure approximately 1 cm3. When this tumor measurement is attained, prepare perioperative therapy reagent.</li>
	<li>Oncolytic virus is one innovative therapeutic that one can administer in the perioperative period. Prepare oncolytic virus (1x109 PFU/1 ml) in 1x sterile PBS and place on ice prior to injection.</li>
	<li>Secure and place mouse into restrainer for intravenous tail vein injection.</li>
	<li>Gently heat mouse tail in warm tap water to visualize lateral tail veins.</li>
	<li>Load a 27 G&#160;&#189; in &#34;Insulin Syringe&#34; with precisely 100 &#956;l of oncolytic virus therapy. Ensure that all air bubbles are removed from syringe column.</li>
	<li>Inject the mouse with 1x108 PFU/100 &#956;l/mouse to one of the lateral tail veins. If the needle is appropriately inserted into a lateral vein, no resistance should be felt when depressing the syringe.</li>
</ol>
5. Complete Resection of Primary Tumor and Abdominal Left Nephrectomy
<ol>
	<li>At 14 days post-tumor cell injection, initiate routine perioperative care following University of Ottawa Animal Care and Veterinary Service approved protocols. Treat mice subcutaneously with Buprenorphine (0.05 mg/kg) 1 hr&#160;before surgery for pain management. Induce and maintain anesthesia using 2.5% isoflurane during the surgery.&#160;Under sterilized conditions and using sterile surgical instruments, surgical site is shaved and scrubbed. Animals are administered subcutaneous fluids and eye lubricant prior to surgery.</li>
	<li>Make a small incision (1-2 cm in length) to gently and completely remove the primary 4T1 tumor from the mammary fat pad.</li>
	<li>Close the incision with 2-3 9 mm staples.</li>
	<li>Expose the abdomen by cutting through the skin and subcutaneous layer along the ventral midline of the mouse.</li>
	<li>Make an incision at the linea alba (3-4 cm) to access mouse peritoneum.</li>
	<li>Expose the left interior side of the abdomen by moving the overlying bowels to the side. Make sure the intestines are kept moist with a saline soaked sterile gauze.</li>
	<li>Using a blunt pair of surgical forceps, grasp the left kidney gently.</li>
	<li>Using a 3-0 wax coated braided silk suture tied into a loop, ligate the hilum of the left kidney and secure with 3 surgical knots. Remove the left kidney with surgical scissors.</li>
	<li>Inspect the suture tie carefully to ensure that adequate hemostasis is achieved.</li>
	<li>Close the subcutaneous layer with a continuous loop suture using 5-0 braided absorbable, then staple skin layer using 9 mm staples. Steps 5.2 to 5.10 should require 10 min/animal.</li>
	<li>Maintain Buprenorphine (0.05 mg/kg) for pain management administered s.c. every 8 hr for 2 days.</li>
</ol>
6. Euthanizing Mice and Processing and Quantification of Lung Tumor Burden
<ol>
	<li>At 28 days post 4T1 tumor injection, euthanize mice according to Animal Care and Veterinary Services protocol at The University of Ottawa.</li>
	<li>Spray down mouse with 70% ethanol.</li>
	<li>Make initial incision with scissors just below the rib cage.</li>
	<li>Expose the thorax by cutting through the skin and subcutaneous layer along the ventral midline of the chest cavity of the mouse.</li>
	<li>Make lateral incisions through skin and tissue on each side up to the neck of the mouse.</li>
	<li>Dissect out the lungs by gently grasping the lung while snipping away the connective tissue above and below the lungs.</li>
	<li>Presoak extracted lungs in cold PBS to remove residual blood, then place in 10% Buffered Formalin.</li>
	<li>Photograph each lung for tumor metastases assessment. Weigh lungs for tumor burden quantification.</li>
</ol>

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Surg. , (2012).</li><li>Shakhar, G., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+prophylactic+measures+against+postoperative+immunosuppression:+could+they+reduce+recurrence+rates+in+oncological+patients.">Potential prophylactic measures against postoperative immunosuppression: could they reduce recurrence rates in oncological patients.</a> Ann. Surg. 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The authors declare&#160;no competing financial interests.

Surgical resection is the mainstay of therapy for patients with localized solid malignancies. Even with complete resection, many patients develop a metastatic recurrence and ultimately die of their disease. The immediate postoperative period provides an ideal environment for the formation of cancer metastases, modulated, in large part, by postoperative NK cell suppression. Despite this, it remains a therapeutic window that is largely ignored. There are currently no standard perioperative anti-cancer therapies aimed at preventing postoperative metastases. The current challenge is to identify safe and promising therapies that will activate NK cells in the perioperative period thereby preventing the establishment of micrometastatic disease. These therapies must be rigorously characterized for safety and efficacy in preclinical animal models and then translated into thoughtfully designed clinical trials.
The primary rationale of developing a mouse tumor model of surgical stress is to explore mechanisms of immune suppression and metastatic spread following surgery and to evaluate innovative immunotherapies with potential for future use in cancer patients undergoing surgery to remove the primary tumor. To accomplish this goal, a 4T1 murine mammary carcinoma model coupled with surgical stress was developed. While the 4T1 cell line is a murine &#34;mammary carcinoma&#34;, the reason to use this cell line is the reproducibility of spontaneous metastases that allows us to evaluate the effects of surgical stress in a realistic cancer model. In this context, the actual origin of the malignancy is less important than the metastatic potential and tumor biology. The second crucial component of our model is the development of an animal surgical procedure to closely resemble human cancer surgery. In this animal surgical stress model, the primary 4T1 breast tumor is excised after it reaches 1 cm3. Because cancer surgeries in human patients involve significant immune suppression, an open-abdomen nephrectomy in &#34;surgical stress&#34; treatment groups is additionally performed. As it compares with more conventional tumor resection in humans, the invasive nature of a full left nephrectomy is very comparable to numerous types of surgical treatments for solid malignancies, including surgery for colorectal cancer, ovarian cancer, kidney cancer, pancreatic cancer, lung and esophageal cancer. In addition, we would argue that the profound physiological changes that occur perioperatively, due to laparotomy+nephrectomy, adequately reproduces the overwhelming physiological changes that occur following invasive surgery for most solid malignancies. To control for perioperative factors that could lead to excessive surgical stress and mortality, the duration of anesthesia and surgery and maintain body temperature during the surgery is precisely defined. All these parameters are precisely executed and defined in our surgery protocol to mirror routine perioperative care in human cancer patients.
The timing of the perioperative treatments is an additional critical component to the perioperative rescue model. 4T1 tumor bearing mice have been previously treated with 3 regimens of influenza vaccine: neoadjuvant (given 5 days prior to surgery), perioperative (given on the same day of surgery) and perioperative + multidose (given on the day of surgery, followed by 2 additional doses given 5 days apart). Remarkably, all 3 modes of vaccine treatment significantly decreased lung metastases22. However, influenza vaccine administered perioperatively as a single dose reduced metastases most effectively. Collectively, these experiments highlight the importance of the immediate perioperative period as a narrow therapeutic window to intervene in the metastatic process.
Perioperative use of innovative immunotherapies such as oncolytic virus and vaccines has exclusively been limited to our research group. We demonstrated for the first time that perioperative administration of novel oncolytic ORF and vaccinia viruses can reverse NK cell suppression following surgery in animal models1. More importantly, this rescue of immune function correlates with a reduction in the postoperative formation of metastases. In human studies, postoperative cancer surgery patients had reduced NK cell cytotoxicity and perioperative OV markedly increases NK cell activity in cancer patients1. Using commercially available prophylactic vaccines, we demonstrated that perioperative influenza vaccine administration significantly reduced tumor metastases and improved NK cell cytotoxicity in preclinical tumor models. In human studies, influenza vaccine significantly enhanced NK cell activity in healthy human donors and cancer surgery patients22.
Many approaches are used to reduce cancer recurrence, including chemotherapy, and radiation, but these therapies are usually administered weeks to months before (neoadjuvant) or after (adjuvant) surgery. Research in this field shows that the immediate postoperative period is critical in determining long-term tumor recurrence rates. Therefore, clinical interventions in the form of immunotherapies during this critical period may have substantial long-term benefits. Our studies using a mouse model of spontaneous lung metastasis and surgical stress provide a unique opportunity to explore novel therapies and determine their potential to prevent perioperative immunosuppression in cancer surgery patients and thereby also reduce cancer recurrence rates.

Surgery is a critical component in the curative cancer treatment of solid tumors, but despite complete resection, many patients develop a metastatic recurrence and ultimately die of their disease. Increasingly, the postoperative period, as a result of the physiologic stress response to surgery, including blood coagulation, release of growth factors, and immune suppression, is recognized as critical time for the development of metastases. Our group1,2 and others3-5 have shown that the immediate postoperative period is a uniquely susceptible time for the formation of metastases.
One of the key mechanisms responsible for the prometastatic effects of surgery is postoperative dysfunction of Natural Killer (NK) cells1-3. NK cells are cytotoxic lymphocytes of the innate immune system involved in the control of tumor growth and metastases6. NK cell dysfunction following surgery has been documented in both human patients1,7-9 and animal models1,10,11. Postoperative NK cell suppression correlates with increased metastases in animal models of spontaneous and implanted metastases1,11-14, while in human studies, low NK activity during the perioperative period is associated with a higher rate of cancer recurrence and mortality15,16.
Despite this, there are currently no cancer therapies specifically addressing the prometastatic changes that occur immediately following cancer surgery. The perioperative period represents a therapeutic window of opportunity in which to intervene in the metastatic process. While traditional cancer therapies, such as cytotoxic chemotherapy, are considered too toxic to be administered to patients recovering from major surgery17, immune therapies are ideal candidates for perioperative administration. Perioperative use of recombinant IL-2 and IFN-&#206;&#179; have been explored in early phase clinical trials demonstrating their potential to prevent postoperative NK cell suppression and improve progression-free survival18-21. Unfortunately, further development has been hindered by tolerability of this nonspecific cytokine therapy combined with major surgery17. Viruses are also potent nonspecific activators of NK cells. Our research has previously demonstrated that preoperative administration of replicating viruses, such as novel anti-cancer Oncolytic Viruses (OV), and nonreplicating viral vaccines, such as influenza vaccine, can inhibit surgery-induced NK cell dysfunction and attenuate metastatic disease1,22.
The surgical model described in this paper has facilitated our understanding of the mechanisms involved in the spread and growth of tumor cells after surgery and allowed us to explore novel targeted therapies that can be administered in the perioperative period. To accomplish this goal, an animal model of surgical stress and spontaneous metastasis wad developed. The model makes use of mouse breast carcinoma tumors (BALB/c - 4T1) that are able to spontaneously metastasize from the primary mammary gland to multiple distant sites in particular the lungs. At day 0, breast cancer cells are implanted in the mouse breast fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules in surgically stressed and no surgery control mice compared are isolated and quantified. In this animal model of cancer and surgery, the effects of surgery on cancer metastases are being studied and the efficacy of perioperative administration of innovative immunotherapies, including replicating and nonreplicating virally based immune- stimulants are tested for the first time.

<table border="0" cellpadding="0" cellspacing="0" width="647">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:111px;">Corning Cell-Gro</td>
			<td style="width:153px;">10-013-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:111px;">HyClone</td>
			<td style="width:153px;">SH30396.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:153px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1X sterile Phosphate Buffered Saline (PBS)</td>
			<td style="width:111px;">Corning cell gro</td>
			<td style="width:153px;">21-031-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Buprenorphine</td>
			<td style="width:111px;">Chiron, Guelph</td>
			<td style="width:153px;">RXN309968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isofluorane</td>
			<td style="width:111px;">Baxter Corp</td>
			<td>1001936040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1/2 ultra-fine syringe</td>
			<td style="width:111px;">Terumo</td>
			<td>30 Gauge, SS05M3009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">9mm staples</td>
			<td style="width:111px;">Braintree</td>
			<td style="width:153px;">ACS BX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-0 braided absorbable suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">UL-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">3-0 wax braided silk suture</td>
			<td style="width:111px;">Covidien</td>
			<td style="width:153px;">S-194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Formalin</td>
			<td style="width:111px;">Fisher</td>
			<td style="width:153px;">SF100-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4T1 tumor cells</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:153px;">CRL-2539</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BALB/c</td>
			<td style="width:111px;">Charles Rivers Labs</td>
			<td style="width:153px;">Strain Code:028</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse tumor model of surgical stress to explore the mechanisms of postoperative immunosuppression and evaluate novel perioperative immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A reproducible mouse model of surgical stress that results in the dramatic enhancement of pulmonary metastases has been developed. At day 28 post 4T1 tumor inoculation (and 14 days post tumor resection +/- abdominal nephrectomy), lungs were harvested and visualized for metastases. Surgery clearly increases the amount of pulmonary metastases compared to untreated mice as demonstrated by lung photographs (Figure 1A), enumeration of lung nodules (Figure 1B) and lung weight (Figure 1C). Preoperative administration of replicating oncolytic virus and inactivated influenza vaccine significantly rescues the prometastatic effects of cancer surgery (Figures 1A-C).
To determine whether NK cells play a mediating role in preventing metastases post-vaccine treatment, NK cells were pharmacologically depleted using anti-asialo-GM-1 in the tumor metastasis model. In the absence of NK cells, we observed an abrogation of the therapeutic effect of perioperative immunotherapies (Figures 2A and 2B). This data suggests that tumor metastases removal in our surgical stress model is mainly mediated through oncolytic virus and influenza vaccine activation of NK cells and subsequent NK mediated tumor lysis. To further characterize NK cell function following perioperative administration of oncolytic virus and influenza vaccine, ex vivo NK cell killing was assessed. Briefly, pooled and sorted DX5+ NK cells were isolated from splenocytes of surgically stressed and control mice. They were cocultured for 4 hr with chromium labeled YAC-1 target cells, at different Effector to Target ratios, followed by measurement of supernatant chromium release with a gamma counter. A significant surgery induced defect in NK cell cytotoxicity along with a significant recovery of NK killing following perioperative administration of oncolytic virus and influenza vaccine compared to surgery alone (Figures 2C and 2D) was observed. Taken together, these results demonstrate that perioperative NK cell suppression can be successfully treated and metastatic disease reduced with novel immunostimulatory therapies.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51253/51253fig1highres.jpg" src="/files/ftp_upload/51253/51253fig1.jpg" /> 
Figure 1. Novel anti-cancer oncolytic virus and influenza vaccine as perioperative therapy against surgery-induced enhancement of lung metastases. Assessment of 4T1 lung tumor metastases at day 28 of indicated treatment groups by (A) photographs of representative lungs, (B) enumeration of lung tumor nodules and (C) lung weights. Data are representative of 3 similar experiments with n=5-10/group (*, p =0.01;**, p &#60;0.0001; n.s., not significant). <a href="https://www.jove.com/files/ftp_upload/51253/51253fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51253/51253fig2highres.jpg" src="/files/ftp_upload/51253/51253fig2.jpg" /> 
Figure 2. Surgical stress increases lung tumor metastases by impairing NK cells. (A,B) Quantification of lung tumor metastases in surgically stressed mice treated with novel perioperative therapy. (C,D) The ability of purified DX5+ NK cells from surgically stressed and untreated controls to kill tumor cells. The data are displayed as the mean percent (+/- SD) of chromium release from triplicate wells for the indicated E:T ratios. Data are representative of 3 similar experiments where n=4-5/group (*, p =0.01;**, p &#60;0.005; n.s., not significant). <a href="https://www.jove.com/files/ftp_upload/51253/51253fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies at JoVE.c

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

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29.8K Views.  Ottawa Hospital Research Institute. A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Video: A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson's Disease

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our understanding of the mechanisms underlying parkinsonian symptoms, since it recapitulates the changes in basal ganglia circuitry and pharmacology observed in parkinsonian patients1-4. However, the precise cellular and molecular changes occurring at cortico-striatal synapses of the output pathways within the striatum, which is the major input region of the basal ganglia remain elusive, and this is believed to be site where pathological abnormalities underlying parkinsonian symptoms arise3,5.
In PD, understanding the mechanisms underlying changes in basal ganglia circuitry following degeneration of the nigro-striatal pathway has been greatly advanced by the development of bacterial artificial chromosome (BAC) mice over-expressing green fluorescent proteins driven by promoters specific for the two striatal output pathways (direct pathway: eGFP-D1; indirect pathway: eGFP-D2 and eGFP-A2a)8, allowing them to be studied in isolation. For example, recent studies have suggested that there are pathological changes in synaptic plasticity in parkinsonian mice9,10. However, these studies utilised juvenile mice and acute models of parkinsonism. It is unclear whether the changes described in adult rats with stable 6-OHDA lesions also occur in these models. Other groups have attempted to generate a stable unilaterally-lesioned 6-OHDA adult mouse model of PD by lesioning the medial forebrain bundle (MFB), unfortunately, the mortality rate in this study was extremely high, with only 14% surviving the surgery for 21 days or longer11. More recent studies have generated intra-nigral lesions with both a low mortality rate &gt;80% loss of dopaminergic neurons, however expression of L-DOPA induced dyskinesia11,12,13,14 was variable in these studies. Another well established mouse model of PD is the MPTP-lesioned mouse15. Whilst this model has proven useful in the assessment of potential neuroprotective agents16, it is less suitable for understanding mechanisms underlying symptoms of PD, as this model often fails to induce motor deficits, and shows a wide variability in the extent of lesion17, 18.
Here we have developed a stable unilateral 6-OHDA-lesioned mouse model of PD by direct administration of 6-OHDA into the MFB, which consistently causes &gt;95% loss of striatal dopamine (as measured by HPLC), as well as producing the behavioural imbalances observed in the well characterised unilateral 6-OHDA-lesioned rat model of PD. This newly developed mouse model of PD will prove a valuable tool in understanding the mechanisms underlying generation of parkinsonian symptoms.

Development of a Unilaterally-lesioned 6-OHDA Mouse Model of Parkinson&#039;s Disease

A protocol for performing unilateral 6-OHDA lesions of the medial forebrain bundle in mice is described. This method has a low mortality rate (13.3 %) with 89% of the surviving animals showing &#62;95% loss of striatal dopamine and 90.63&#177;-4.02 % ipsiversive rotational bias towards the side of the lesion.

The unilaterally lesioned 6-hyroxydopamine (6-OHDA)-lesioned rat model of Parkinson's disease (PD) has proved to be invaluable in advancing our ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...